Abstract: Background and Objective: Chlorophyll mutants are used as markers in genetic, physiological and biochemical investigations. The selection of effective and efficient mutagen(s) is very essential to recover high frequency and spectrum of desirable mutations. Present investigation was undertaken to understand the response of lentil variety Pant L-406 to various chemical mutagens for identifying such mutagenic-treatment causing maximum chlorophyll mutations which could eventually be exploited for inducing viable and economically useful mutations in subsequent generations. Materials and Methods: Seeds of lentil were treated with chemical mutagens namely methylmethane sulphonate (MMS), hydrazine hydrate (HZ) and sodium azide (SA) for 6 h. Three replications of 100 seeds per treatment were sown in complete randomized block design (CRBD) to raise M1 generation. The M1 plants were harvested separately and seeds sown in next season in plant progeny rows to raise M2 generation. Chlorophyll mutations were observed in M2, when seedlings were 8-15 days old. The frequency of chlorophyll mutations, mutagenic effectiveness and efficiency were determined as per the standard formulae. Results: Different types of chlorophyll mutants viz., albina, xantha, chlorina, maculata and virescent were observed in the field when seedlings were 8-15 days old. All these chlorophyll deficient mutants were lethal except maculata and virescent which produced few seeds at maturity. The MMS treatments induced the highest frequency of chlorophyll mutations followed by HZ and SA. The frequency of chlorophyll mutations was dose dependent and increased with increasing concentrations of the mutagens. ‘Chlorina’ followed by ‘xantha’ outnumbered the other types of chlorophyll mutants in all the mutagens. Higher effectiveness was recorded with HZ treatments, whereas SA was found to be the most efficient mutagen compared to HZ and MMS. Conclusion: Chlorophyll mutations, although do not have much economic importance due to their lethal nature, such a study could however, be useful in identifying the threshold dose of a mutagen that would increase the genetic variability and number of economically useful mutants in subsequent generations.
INTRODUCTION
Lentil (Lens culinaris Medik) is one of the most important legume crops worldwide including India. India is the largest producer of lentil and contributes to about 32% of total lentil production in world. Of late, due to stagnation and decreasing production and rapid increase in population, the net availability of total pulses has come down from 60 g/day/capita in 1951 to 37 g/day/capita now although India ranked 23rd in lentil productivity1,2. Besides stubble, which is also rich in many valuable components, the grains are composed of 60% of carbohydrates, 26% protein, 7.5% of iron, 2% of sugars and 0.87% of thiamine (vitamin B1) which are essential ingredients of both human and animal feeds3,2.
Mutation breeding has been considered as one of the gizmo of atomic age and a much promising breeding short cut which could be beneficially utilized for tailoring the desired varieties of different crop plants. The major advantage of the use of induced mutations is the possibility to correct one or few negative characters of a cultivar, without changing the major parts of its total genetic set up. Mutation breeding has now become a novel and eco-friendly but useful and in some situations even an indispensable tool which can be employed by the plant breeders4.
The enhancement of mutation frequency and alteration of mutation spectrum in a predictable manner are the two important goals of mutation research. In past, varied approaches have been tried to achieve these goals5-7. Chlorophyll mutations are considered as the most dependable indices for evaluating the genetic effects of mutagenic treatments and are also used as genetic markers in basic and applied research. Several authors have reported the incidence of different types of chlorophyll mutations in M2 generation following treatments with different mutagenic agents in various crop plants8-20.
Before the start of any sound breeding programme, knowledge of relative biological effectiveness and efficiency of various mutagens and their selection is essential to recover high frequency of desirable mutations21,22. It is not necessary that an effective mutagen shall be an efficient one also23-25. Mutagenic effectiveness relates mutagen dose to the mutational events, while mutagenic efficiency gives an idea of genetic damage in relation to the total biological damage caused in M1 generation26. Though both of these are two different properties but the usefulness of any mutagen in plant breeding programme depends on both of them. The response of biological system to mutagens is influenced to a varying degree by numerous biological, environmental and chemical factors. These factors modify the effectiveness and efficiency of different mutagens greatly and same is true for the mutation rate27-29. The present investigation was undertaken with the aim to study the effects of MMS, HZ and SA on the frequency and spectrum of chlorophyll mutants and to evaluate the relative effectiveness and efficiency of these chemical mutagens in M2 generation of lentil variety Pant L-406.
MATERIALS AND METHODS
A field experiment was conducted during the rabi season of 2006-2007 and 2007-2008 at University Agricultural Farm, Aligarh Muslim University, Aligarh, Uttar Pradesh, India. Dry and healthy seeds of lentil (Lens culinaris Medik) variety Pant L-406, pre-soaked in distilled water for 9 h, were treated with three chemical mutagens viz., methylmethane sulphonate (MMS): A monofunctional alkylating agent manufactured by Sissco Research Laboratories Pvt. Ltd., Mumbai, India, hydrazine hydrate (HZ): A base analogue, manufactured by Qualigens Fine Chemicals, Mumbai, India and sodium azide (SA): A respiratory inhibitor, manufactured by Indian Drugs and Pharmaceuticals Ltd., Hyderabad, India for 6 h. The concentrations used for MMS ranged from 0.1-0.4%, whereas these were 0.01-0.04% for HZ and SA. The solutions of MMS and HZ were prepared in phosphate buffers of pH 7, whereas SA solution was prepared in phosphate buffer adjusted to pH 3. To facilitate uniform absorption, large quantities of the solution of mutagens, approximately three times the volume of seeds were used26. Following these treatments, the seeds were thoroughly washed in running tap water to remove the residue mutagens from the seed surface. The treated seeds were directly sown in the field along with untreated control. Three replications of 100 seeds/treatment were sown in complete randomized block design (CRBD) to raise M1 generation. The M1 plants were harvested separately and the seeds sown the next season in plant progeny rows to raise M2 generation. The distance between the seeds in a row and between the rows was kept at 30 and 60 cm, respectively.
Chlorophyll mutations were scored in M2 generation when seedlings were 8-15 days old. They were identified and classified according to Gustafsson30. The frequency of chlorophyll mutations was calculated as per Konzak et al.26:
Mutagenic effectiveness is a measure of the frequency of mutations induced by unit dose of a mutagen (time×concentration), while mutagenic efficiency represents the proportion of mutations in relation to biological damage. Formulae suggested by Konzak et al.26 were used to evaluate the mutagenic effectiveness and efficiency of the mutagens:
*Biological damage: For measuring the biological damage, three different criteria were used:
| • | Injury: i.e., percentage reduction in seedling height (Mp/I) |
| • | Sterility: i.e., percentage reduction in pollen fertility (Mp/S) |
| • | Inhibition: i.e., percentage inhibition in seed germination (Mp/R) |
RESULTS AND DISCUSSION
Chlorophyll mutation frequency in M2 generation is one of the most reliable measures for evaluating the mutagen-induced genetic alterations. Based on the intensity of pigmentation at the seedling stage, 5 different types of chlorophyll mutants viz., albina, xantha, chlorina, maculata and virescent were recorded in segregating M2 plants of lentil (Table 1). Most of the chlorophyll mutant types (albina, xantha and chlorina) were lethal and survived to seedling stage only. Maculata and virescent types however survived upto maturity and produced few seeds. A brief description of different chlorophyll mutants is given below:
| • | Albina: Lethal mutant, white leaves of seedlings, survived for 8-10 days after germination |
| • | Xantha: Yellow colored leaves, seedlings survived for 10-15 days only |
| • | Chlorina: Light green first pair of leaves in seedlings, emerging leaves were also light green and became darker with the approach of maturity, seedlings died within 15 days |
| • | Maculata: Yellow or whitish dots on leaves. Plants were vigorous in growth, late in maturity and produced few seeds |
| • | Virescent: White or light yellow colored leaves with patches of yellowish green or green color. Mostly, yellowish or light green patches completely disappeared and the normal color was regained. They were as vigorous as the normal plants and set seeds |
Of the five types of chlorophyll mutants recorded in M2 generation, ‘chlorina’ followed by ‘xantha’ types were predominant in all the three mutagens. Joshi-Saha et al.19 reported that electron beam and gamma irradiation induced more number of ‘chlorina’ mutations in variety vijay of chickpea, while Khan et al.25 reported that chemical mutagens induced high frequency of ‘chlorina’ mutants in M2 generation of chickpea. Occurrences of ‘chlorina’ mutants in large number of crops have been attributed to different causes such as impaired chlorophyll biosynthesis, degradation of chlorophyll and deficiency of carotenoids31.
Among the mutagens used in the present study, MMS induced the higher frequency and wider spectrum of chlorophyll mutations. The high incidence of chlorophyll mutations induced by MMS, in the present study, may be due to its specificity to affect certain regions of chromosomes.
| Table 1: | Frequency and spectrum of chlorophyll mutants in M2 generation of lentil |
| MMS: Methylmethane sulphonate, HZ: Hydrazine hydrate, SA: Sodium azide | |
| Table 2: | Effectiveness and efficiency of the mutagens in M2 generation of lentil |
| MMS: Methylmethane sulphonate, HZ: Hydrazine hydrate, SA: Sodium azide | |
Chlorophyll development seems to be controlled by many genes located on several chromosomes which could be adjacent to centromere and proximal segment of chromosome32-33. Mutations in these chlorophyll genes are reflected in M2 and subsequent generations in the form of different types of mutants. A linear relationship between chlorophyll mutations frequency and dose was observed with all the three mutagens (Table 1). Similar dose dependent increase in chlorophyll mutation frequency was reported by Kharkwal9 and Bara et al.34 in chickpea, Arora and Kaul35 in pea, John36 in cowpea, Das and Kundagrami37 in grasspea, Wani et al.38 in mungbean and Patil and Rane39 in cluster bean. The frequency of chlorophyll mutations induced by SA was less as compared to MMS and HZ. The low chlorophyll mutations frequency in SA treatments may be due to the inhibition of catalase and peroxidase and an increase in peroxide concentration in the cells40.
Mutagenic effectiveness and efficiency: Data on effectiveness and efficiency of various mutagenic treatments calculated on the basis of frequency of chlorophyll mutations is given in Table 2. It was found that effectiveness and efficiency were higher at lower concentrations of all the three mutagens used. This is because the lower dose may cause relatively less damage enabling the organism to express the induced gene mutations successfully. The estimates of effectiveness ranged from 24.42-66.66 in MMS treatments, whereas the effectiveness of HZ and SA treatments ranged from 223.20-654.66 and 194.41-595.16, respectively. Mutagenic effectiveness decreased at the highest concentration of all the three mutagens. The order of mutagens based upon effectiveness was HZ>SA>MMS. The HZ was also reported to be an effective mutagen in maize41, barley42, sorghum43 and mungbean38.
Mutagenic efficiency varied depending upon the criteria selected for its estimation. In this study, the mutagenic efficiency, worked out on the basis of seedling injury (Mp/I), pollen sterility (Mp/S) and inhibition in seed germination (Mp/R), showed a decline with increasing concentrations of all the treatments (Table 2). These results are in conformity to those reported earlier by Khan and Siddiqui44. The greater efficiency at lower doses is because the biological damage (seedling injury, pollen sterility and inhibition in seed germination) generally increased with the enhancement in the dose at a higher rate than the mutations yielded in M2 at the same dose26. The mutagenic efficiency was more pronounced in SA treated population than those treated with HZ and MMS. Further, the efficiency calculated on the basis of inhibition in seed germination was higher as compared to those based on pollen sterility and seedling injury. These results are in agreement to the earlier findings of Wani and Khan45.
CONCLUSION
Improvement in frequency and spectrum of mutations in predictable manner and thereby achieving desired plant characteristics for their direct or indirect exploitation in breeding programmes is an important goal of mutation research. Occurrence of chlorophyll deficient mutants is attributed to change in gene or set of genes responsible for chlorophyll mutations. Mutations in these genes are reflected in M2 and subsequent generations in the form of different chlorophyll mutants. Chlorophyll mutants though do not have much economic importance due to their lethal nature, yet they could be useful in identifying the threshold dose of a mutagen that would increase the genetic variability and number of economically useful mutants in later generations.
SIGNIFICANCE STATEMENTS
This investigation was aimed to study the frequency of chlorophyll mutations and determine the effectiveness and efficiency of different mutagens in mutant (M2) generation of lentil. Alteration in the nucleotide sequence of genes controlling the synthesis of enzymes required for biosynthesis of pigments lead to chlorophyll mutations. Chlorophyll mutations are important for identifying gene function and elucidating the chlorophyll metabolism and its regulation. In this study, the chlorophyll mutants induced by different chemical mutagens could be utilized in different mutation breeding programmes for inducing viable mutations of greater economic and agronomic importance.
ACKNOWLEDGMENTS
The author is grateful to the Chairman, Department of Botany, Aligarh Muslim University, Aligarh, India and Principal, Government Degree College (Boys), Anantnag, Jammu and Kashmir, India for providing necessary research and laboratory facilities.