Induced genetic variability for yield and yield traits in aromatic rice ( Oryza sativa L . )

The nature of induced mutation for polygenic variability was studied in two traditional aromatic rice genotypes, viz., Kalanamak and Badshah Bhog. Induced variability was observed in both the M2 and M3 generations indicated the possible selection for quantitative characters. The shift in mean was not found to be unidirectional nor equally in both directions in all the treatments. Most of the mutagenic families in different treatments showed shift in mean toward negative side coupled with high range and CV as compared to their respective control. In general, combination treatments of 30kR gamma-ray+ 0.2% EMS and 40 kR gamma-ray+ 0.2% EMS induced maximum variability for most of the traits. The use of physical and chemical mutagens or a combination of both has been an important tool for the increase of variability in agronomic traits. In general, there was reduction in variability, as judged from range and CV, in M3 as compared to M2 in all the treatments and traits in both the genotypes. The present investigation had clearly demonstrated the high potentials of 40 kR gamma-ray +0.2% EMS followed by 40 kR gamma-ray and 30 kR gamma-ray +0.2% EMS were found to be more useful in releasing desirable variability for yield and component traits in desired directions for most of the characters in both the genotypes in both the generations.


INTRODUCTION
India has largest area under paddy in the world and ranks second in the production after China (Anonymous, 2007).The primary objective of the mutation breeding is to enhance the frequency and spectrum of mutations and also to increase the incidence of viable mutations.Since genetic variability is a pre-requisite for any successful breeding programme, the creation and management of genetic variability becomes central base for crop improvement programme.Rice is a diploid and selfpollinated crop and it possess enormous possibilities of improvement through mutation breeding; significant achievements have been made in developing new rice varieties with desirable characters through mutation breeding (Baloch et al., 2002).The present investigation has been taken up with two genotypes of rice which included Kalanamak (Medium Slender Grain) and Badshah Bhog (Short and flattened grain).

MATERIALS AND METHODS
Two thousand pure, healthy and dry seeds (moisture, 12%) of the two rice varieties, namely, Kalanamak and Badshah Bhog were irradiated with 10, 20, 30 and 40 kR doses of 60 Co. gamma rays at National Botanical Research Institute, Lucknow, Uttar Pradesh.Irradiated and unirradiated seed lots of each variety were divided into two equal parts (one thousand each).First lot was used as gamma rays treatment alone and other for combined treatment of gamma rays + Ethyl methane sulphonate (0.2%) and EMS (0.2%) alone.For chemical mutagen treatment, seeds were submerged for six hours in distilled water to insure complete hydration of the seeds at 30°C in incubator.Soaked seeds were blotted for removing surface water before transferring them into Ethyl methane sulphonate (0.2%) prepared with phosphate buffer solution having the pH 7.0 for a period of 6 h. in incubator (25°C) and were given intermittent shaking throughout the period of treatment to maintain uniform concentration.After EMS treatment, the seeds were thoroughly washed in running tap water for one hr to remove residual chemicals.
For micro mutational studies, all the M 1 plants having 60% pollen fertility or more (minimum being twenty plants per treatment) were advanced to raise M 2 generation following the procedure adapted by Gaul (1964).Seeds of twenty M 1 plants, selected on the basis of pollen fertility as described above, were sown separately in the nursery during rainy season of 2006.Twenty one days old seedlings of all the twenty M 1 plants selected from each treatment were transplanted in the well puddle field in Randomized Block Design with three replications.Agronomic practices were the same to that of M 1 generation.Data on five competitive normal looking plants from each M 2 families were taken randomly to record the observations on nine quantitative characters, namely, days to 50% flowering, days to maturity, plant height (cm), panicle length (cm), number of panicle bearing tillers/plant, number of grains/panicle, 100-seed weight (g) and grain yield/plant (g).Since the minimum number of promising micro-mutants in M 2 families for any one of the treatments was twenty, 5 normal looking plants were selected at random from each of these 20 families.Promising micro-mutants exhibiting higher grain yield from the family coupled with higher coefficient variation compared to their respective control were selected to grow M 3 generation treatment-wise.Since numbers of micro-mutations were variable in each treatment, only top twenty micro mutants were sown on raised nursery beds during the rainy (Kharif) season 2007.The 21 days old seedling of all the twenty M 2 plant progenies were transplanted in well puddle field at the distance of 20 × 10 cm from row to row and plant to plant, respectively as has been done in M 2 generation.Randomized Block Design with three replications was followed for transplanting.Each micro mutant was transplanted in three rows of 4 m in length.Recommended agronomic practices were followed to raise good crop.The quantitative and quality traits studied in the M 2 were also studied in the M 3 on five normal looking plants selected at random from each mutant families in each treatment in both the varieties.

RESULTS AND DISCUSSION
The primary objective of the mutation breeding is to enhance the frequency and spectrum of mutations and also to increase the incidence of viable mutations.Many physical and chemical mutagens have been used for inducing viable mutants in rice.In mutation breeding the choice of the mutagen is most important, and various methods have been developed to ascertain the efficiency of mutagen(s) and mutagenic treatments for the induction of desirable characters in a cultivated crop.Since genetic variability is a pre-requisite for any successful breeding programme, the creation and management of genetic variability becomes central base for crop improvement programme.Selection and hybridization are conventional methods for improvement of qualitative and quantitative traits.In this context, it is quite desirable to opt for induced mutagenesis which is recognized as a quick and successful method in creating genetic variability and bringing about desirable improvement.Mutations may be artificially induced by a treatment with certain physical or chemical agents; such mutations are known as induced mutations.

Plant height
In Kalanamak and Badshah Bhog (Table 1 and Figure 1), all but 10 kR, EMS, and 10 kR gamma-ray+ EMS treatments showed significant reduction in mean values coupled with high variability, as evident from range and CV in both the M 2 and M 3 generations.Remarkably, treatment with 30 kR gamma-ray caused shift in mean towards positive side in both the generations as compared to the control.The magnitude of variability, as judged from range towards desirable side (negative shift in mean values), was very high at 40 kR gamma-ray and 40 kR gamma-ray+ EMS in both the generations.Induction of mutants with short plant height in the treatments with 40 kR gamma-ray and 40 kR gamma-ray +EMS were very remarkable in both the generations.In Badshah Bhog, all the treatments except 10 and 20 kR gamma-ray alone and their combination treatments with EMS caused significant reduction in plant height in both M 2 and M 3 generations.The shift in mean towards negative direction that is, towards short stature was more evident in the treatments 30 kR gamma-ray+ EMS and 40 kR gamma-ray, as judged from the lower values of range in both the generations.EMS treatment significantly increased the mean values (shift towards positive side) for plant height in both the generations.

Days to 50% flowering
In Kalanamak and Badshah Bhog (Table 2 and Figure 2), all but 10, 20 and 10 kR gamma-ray + EMS treatments showed significant decline in mean values coupled with high variability, as evident form range and CV, in both M 2 and M 3 generations.The combination treatments produced more variability as compared to single treatments.The magnitude of variability, as judged from range, towards desirable side (negative shift in mean) was very high in treatments 20 kR gamma-ray +EMS

Days to maturity
In Kalanamak and Badshah Bhog (Table 3 and Figure 3), all but treatments `10, 20 and 10 kR gamma-ray + EMS showed significant reduction in days to maturity in both the generations in the genotype Kalanamak.The shifts toward negative side that is, early maturing were more pronounced in the treatment 40 kR gamma-ray + EMS as evident from the lower value of the range.While in other genotype, Badshah Bhog all the treatments except 10 kR gamma-ray, 30 kR gamma-ray and 10kR gamma-ray + EMS caused significant reduction in mean days to maturity in both the generations.Early maturing mutants were noted in the treatments 40kR gamma-ray + EMS, 30 kR gamma-ray + EMS and 40 kR gamma-ray.It was remarkable to note the induction of very early maturing (130-138) mutants in Kalanamak as compared to Badshah Bhog (induction of early mutants with 140-145 days).Single treatment with EMS alone caused significant positive shift in mean days to maturity in both genotypes and generations.

Panicle length
All but treatments 10 kR gamma-ray, 20 kR gamma-ray, EMS and 10 kR gamma-ray + EMS showed significant reduction in panicle length in both the generations in the genotype Kalanamak (Table 4 and Figure 4).The significant shift in the mean values towards positive side for panicle length was noted in treatment 30 kR gammaray in both the generations.From the upper values of range, treatments 40 kR gamma-ray, 30 kR gamma-ray + EMS and 40 kR gamma-ray + EMS were important in the M 2, while in the M 3 only treatment 40 kR gamma-ray + EMS was remarkable.In Badshah Bhog, all the treatments except 10, 20 and 10 kR gamma-ray +EMS caused significant reduction in mean values for panicle length in both the generations.The significant shift in mean in positive side in panicle length was noted in the treatment with EMS in both M 2 and M 3 generations, while 30kR gamma-ray caused significant increase in mean values in M 2 generation only.

Panicle bearing tillers per plant
In Kalanamak (Table 5 and Figure 5), all but 10 kR gamma-ray, 20 kR gamma-ray, EMS and 10 kR gammaray + EMS treatments showed significant decline in mean values from the control coupled with high variability, as evident from the range and CV, in both M 2 and M 3 generations.The magnitude of variability, as judged from the range towards desirable side (positive shift in mean), was very high in 40 kR gamma-ray +EMS in both the generations.Induction of mutants in the treatment with 40 kR gamma-ray +EMS was remarkable in M 2 generation in the genotype Kalanamak.In Badshah Bhog (Table 5 and Figure 5), all the treatments except 10 kR gamma-ray, EMS, 10 kR gamma-ray + EMS and 30 kR gamma-ray + EMS caused significant reduction in panicle bearing tillers per plant in both M 2 and M 3 generations.The shift in mean toward positive direction that is, higher panicle bearing tillers per plant was noted in treatment with 30 kR gamma-ray in the both M 2 and M 3 .The mutants with increased panicle bearing tillers per plant were noted in most of the treatments in both the generations.

Number of grains per panicle
In Kalanamak (Table 6 and Figure 6), all but 10 kR gamma-ray, EMS and 10 kR gamma-ray + EMS treatments showed significant shift in mean values of number of grains per panicle as compared to control coupled with high variability, as evident from the range and CV, in both M 2 and M 3 generations.The treatments 20 kR gamma-ray and 30 kR gamma-ray + EMS induced significant shift in mean toward positive side in both the M 2 and M 3 .Looking at higher values of range, combined treatments 30 kR gamma-ray + EMS and 40 kR gammaray + EMS were most promising, in both the generations as compared to control .Drastic effects of the treatments as judged from lower values of range, were very high in all the treatment as compared to control in both the generations.In Badshah Bhog (Table 6 and Figure 6), all the treatments except 10 kR gamma-ray, EMS and 20 kR gamma-ray + EMS in M 2 and 10 kR gamma-ray, EMS, 10 kR gamma-ray + EMS and 20 kR gamma-ray + EMS in M 3 showed significant shift in mean.The positive shift in mean was noted only in treatment 30kR gamma-ray in both M 2 and M 3 generations.Combined treatments (30 kR gamma-ray + EMS and 40 kR gamma-ray +EMS) were most promising,        as judged from high CV and isolation of mutants with increased number of grains per panicle as compared to control in both M 2 and M 3 generations.

100-seed weight
The significant shift in mean values as compared to control coupled with high variability, as evident from the range and CV, in both M 2 and M 3 generations were noted in Kalanamak (Table 7 and Figure 7), all the treatments except 10 kR gamma-ray, 20 kR gamma-ray, EMS and 10kR gamma-ray + EMS.While positive shift in mean was noted only in treatment 20 kR gamma-ray + EMS in both the M 2 and M 3 .Mutants with high seed weight as compared to control were noted in all the treatments except 10 kR gamma-ray, 20 kR gamma-ray, EMS, and 10 kR gamma-ray + EMS in both the generations.In Badshah Bhog (Table 7 and Figure 7), all the treatments except 10kR gamma-ray, EMS, 10 kR gamma-ray + EMS and 20 kR gamma-ray +EMS caused significant shift in mean 100-seed weight in M 2 and M 3 generations.The shift in mean toward positive direction i.e., higher 100seed weight was noted in treatment with 30 kR gammaray in both M 2 and M 3 and 20 kR gamma-ray + EMS in only M 3 .The mutants with increased 100-seed weight were noted in treatments 40 kR gamma-ray and 40 kR gamma-ray + EMS in both the M 2 and M 3 , while in treatment 30 kR gamma-ray + EMS in M 3 only.

Grain yield per plant
In Kalanamak (Table 8 and Figure 8), all the treatments except 10 kR gamma-ray, 20 kR gamma-ray, EMS and 10 kR gamma-ray + EMS caused significant shift in mean values as compared to control in both M 2 and M 3 generations; positive shift in mean was noted in the treatments 20 kR gamma-ray in M 3 only and 20 kR gamma-ray + EMS in both M 2 and M 3. It was remarkable to note mutants with increased grain yield per plant as judged from upper values of range, in most of the treatments in both M 2 and M 3 generations.While in Badshah Bhog (Table 8 and Figure 8), all treatments except 10 kR gamma-ray, EMS, 10 kR gamma-ray + EMS and 20 kR gamma-ray +EMS caused significant shift in mean grain yield per plant as compared to control in both the generations; positive shift in mean was noted only in treatment 30 kR gamma-ray.Combined treatments with 30kR gamma-ray and 40 kR gamma-ray were more drastic in reducing grain yield per plant in both the generations in both the genotype, Kalanamak and Badshah Bhog.Mutants with high grain yield as compared to the control, in most the treatments were remarkable to note.The use of physical and chemical mutagens or a combination of both has been an important tool for the increase of variability in agronomic traits (Bansal et al., 1990;Agrawal et al., 2000;Sharma et al., 2008).The potentiality of ionizing radiation and chemical mutagens is different and their ability to induce mutation varies from crop to crop and genotype to genotype.Therefore, it is desirable to have the appropriate treatment schedule before under taking the mutagenesis.
On the other hand, Gupta and Swaminathan (1967) suggested that different M 2 families should be analysed for their mean and variance and families showing superior mean and increased variance over the control should be selected.Similar selection process was applied in the present study and also advocated by various workers in rice as well as other crops (Singh, 2003;Singh and Singh, 2003).In general, mutagenic treatments had resulted in decreased mean coupled with enhanced variability in both genotypes and generations as compared to their respective control, though the magnitude of shift in mean varied with the treatment, genotype and trait.The decline in means of treated population was demonstrated in rice (Jana and Roy, 1973;Awan et al., 1980;Siddiqui and Singh, 2010).
The decrease in means of mutagen treated population might be due to greater frequency of mutations with detrimental effects or due to difference in magnitude of induced individual change.The shift in mean was not found to be unidirectional nor equally in both directions in all the treatments.For days to flowering and maturity and plant height shifts in mean toward negative direction, as also noted in the present case, were of great significance as they yielded high frequency of mutants with early flowering and maturing and short stature.The induction and isolation of short statured with early maturing mutants having other desirable traits of the parental genotypes would be quite rewarding.In rice several workers (Singh and Singh, 2003;Domigo et al., 2007;Siddiqui and Singh, 2010) also found induced variability in desired directions for earliness and short stature.
The unidirectional variability towards positive side for grain yield and its component traits, as noted in the present case, were of great significance.This had yielded large number of mutants with improved grain yield per plant as well as other component traits irrespective of genotypes.Several workers also noted similar results after mutagenic treatment in rice (Singh andSingh, 2003, Siddiqui andSingh, 2010).Auxiliary traits like, panicle bearing tillers/plant, panicle length, number of grains per panicle and 100-seed weight on an average, showed positive shift in mean coupled with enlarged variability towards both sides.These auxiliary traits, as expected because of their positive correlation with grain yield, contributed significantly towards grain yield.Similar results were also noted in several crops, such as, rice (Baloch et al., 2002;Elayaraja et al., 2005;Domingo et al., 2007;Bughio et al., 2007;Siddiqui and Singh, 2010).
Hence, in the present study, most of these auxiliary traits were studied to screen out the high yielding mutants

Table 1 .
Range, mean and coefficient of variation (CV) for Plant height (cm) in M 2 and M 3 generations.

Table 2 .
Range, mean and coefficient of variation (CV) for Days to 50% flowering in M 2 and M 3 generations.

Table 3 .
Range, mean and coefficient of variation (CV) for Days to maturity in M 2 and M 3 generations.

Table 4 .
Range, mean and coefficient of variation (CV) for panicle length in M 2 and M 3 generations.

Table 5 .
Range, mean and coefficient of variation (CV) for panicle bearing tillers per plant in M 2 and M 3 generations.

Table 6 .
Range, mean and coefficient of variation (CV) for Number of grains per panicle in M 2 and M 3 generations.