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EXECUTIVE SUMMARY3. PCR Project Completion Report

OBJECTIVES OF THE PROJECT:-1) To study the effect of physical and chemical mutagen on phenology of plants, 2)To study the spectrum of mutation induced by mutagens, 3) To study yield parameters such as number of primary, secondary branches pod per plants seed storage protein, 4)To study the cytological abnormalities 5)To study the induced mutation in qualitative and quantitative traits which could be utilized directly or introduced into chickpea improvement programme.INTRODUCTIONCereals are most beneficial plants used as food. The term 'cereal' traced back in the Roman history associated with a festive occasion celebrated, in the honor of the Goddess Ceres- a giver of grains, with wheat and barley grains at the time of harvesting.Chickpea is a one of the most important grain legumes of Indian Subcontinent (Wani and Anis, 2008). Chickpea is third most important cool season pulse crop of the world (FAO, 1994). It is important food legume of dry lands and tropics in the world and production worldwide (Toker and Canci, 2003). Chickpea genotype adapted to these condition by acquiring bushy, spreading and indeterminate growth habit and photo-thermo-sensitivity (Bahl et al., 1979).Asia is most important chickpea producer and India is largest single producer of the crop (Gebisa et al., 2000). Chickpea became principle pulse crop and a dietary mainstay in the Indian subcontinent (Muehlbauer, 1993).

The available genetic variability has been exploited in the breeding programme narrowed the genetic base (Wani and Anis, 2008). A few undesirable characters constraints the use of wild Cicer in chickpea breeding programs (Jaiswal et al., 1986). The mutagenesis could create many different mutants alleles with varied and different degree of great modification (Brown, 2003). Mutation breeding could be used towards the induction and improvement of economically important traits and characters as well as elimination of undesirable gene from the elites lines (Lippart et al., 1964).

The genus Cicer belongs to the family leguminoceae containing 9 annuals and 31 perennial species are distributed worldwide, of which Cicer arietinum L. only one of 9 annuals is under cultivation. The Cicer arietinum L .and Cicer reticulatum have 2n=16 chromosomes as in other annuals species (Muehlbauer, 1993).Ledizinsky (1975) collected and identified wild progenitor of chickpea C. reticulatum in Turky. India is the secondary center of genetic diversity (Smartt, 1990a, b).The systematic position of Cicer is as under

Division :- AngiospermClass :- DicotyledonsSubClass :- PolypetalaeSeries :- CalycifloraeOrder :- RosalesFamily :- Papilionaceae (Leguminosae)

Genus :- Cicer

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The rotational cropping pattern with legume crop could serve a basis to break disease cycle in winter thereby improving the soil fertility profile and weed control (Davies et al., 1985). Chickpea breeding programs have limited themselves to a small number of cultivated genotypes having sources of biotic stress resistance and abiotic stress tolerance with little or no use of wild species (Singh et al., 1994).

Chickpea is a good source of carbohydrates and protein with 80% of total dry seed weight. The dryland crops that feed the poor across the semi-arid tropics are receiving far less attention than are the 'glamour crops'. Chickpea is an example - a relatively little-researched crop that is nonetheless an important source of calories and protein for the poor across Asia, the Middle East, and North Africa.

The limited utilization of the wild species in the chickpea breeding programs due to interspecific crossing barriers and deleterious linkage drag. Sources of resistance and tolerance to these constraints exist in the wild Cicer germplasm yet remain largely unused by conventional breeding programs (Muehlbauer et al., 1994; Singh et al., 1994; Erskine et al.,2001; Knight et al., 2002; Buhariwalla et al., 2005).

Mutagenesis was used to develop cultivars with good adaptability to exogenous factors and with increased productivity (Mihov and Mehandjiev, 1982). Seed mutagenesis has been reported to enhance genetic variability of yield parameter in Capsicum annuum (Jabeen and Mirza, 2002) in mungbean (Singh et al., 2001) in green gram (Sharma, 1998) in wild and cultivated urd and mungbean (Ignacimuthu and Babu, 1993).

The number of Chemical substances such as Ethyl Methyl Sulphonate (EMS), Ethylene Imine (EI), Sodium Azide (NaN3) were reported more effective mutagen in mutation breeding (Gottschalk, 1986).The genetic erosion resulted into a bottleneck and various techniques to induce mutations and artificially increase variation emerged in the middle of the last century (Smartt and Simmonds, 1995) The X-ray radiation was used as a mutagen due to its easy availibility to researchers. Muller (1927) showed that X-ray treatment could increase the mutation rate in a Drosophila population by 15,000%. The phenotypic variation in barley seedlings and sterility in maize tassels has been observed after exposure to X-rays and radium(Stadler,1928).

MATERIAL AND METHODThe germplasm of wild chickpea (Cicer reticulatum L.) Accession No. ICC 17164 JM

2106 and ICC 17121 JM 2100 was obtained from ICRISAT, Patancheru (AP) India for the present investigation. The seeds were divided into three sets. The seeds of 1st set treated with three different concentration viz. 0.1%, 0.2%, 0.3%,of Sodium azide (SA) and encoded as T2, T3, T4 respectively. The seeds of 2nd set were treated with combination treatment of SA and X-rays radiation viz. 0.1% SA+5KR, 0.2% SA+10KR, 0.3% and SA +15KR and encoded as T5, T6, T7 respectively. The healthy seeds were first treated with 0.1% to 0.3% SA thereafter washed thoroughly and soaked with blotting paper to remove any residual effect of treating solution then the pre-treated seeds were irradiated with 5KR to 15 KR X rays. The seeds of 3rd set were treated with different doses 5 KR, 10 KR, 15 KR of X-ray radiation and

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encoded as T8, T9, and T10 respectively.While T1 as the untreated control. The treated seeds were subjected for germination in the petriplates lined with two to three layers of moist filter paper. The 3-4 healthy and actively growing root-tips were excised on reaching the length about 1 to 1.5cm during the time interval of 10.00 am to 11.30 am. The slides were prepared by following standard squash technique (Sharma and Sharma, 1990). The 2% aceto-orcein stain was used in the present cytological study. The random 10 counts per slide were scored to cover maximum surface area of the slide for computing the Mitotic Index,standard error for each treatment. Mitotic index was determined for each treatment as Eq. No. (1) (Bhalla et al., 1973).The various mitotic abnormalities were observed in the present study. The data obtained was statistically analyzed as per Eq. No. (2) given by Panse and Sukhatme (1978). The mitotic index is percentage frequency of the dividing cells out of total cells scored. Total No. of dividing cells Mitotic index = -------------------------------x100 ------------------------ (1) Total No. of cells scored ∑ xi

(μ) X = ------------------- --- (2) n

Where, X (μ) - mean, ∑ xi – Sum of ‘i’ observation, n- Number of observation.

All the treated seeds alongwith the untreated control T1 seeds were sown to raise M1 generation in triplicate. The 10 seeds of each treatments were presoaked in distilled water for overnight placed in Petri plates lined with 2-3 layers of moist filter paper and sterile distilled water was added to keep the moisture level proper. The germination percentage and time required for germination were observed (ISTA, 1993). The phenotypic data such as shoot length height, number of branches, number of flower per plant, pod per plants, pod length, grain per pod were recorded at regular interval and presented in Table 5 . The data subjected to the statistical analysis and computation of various quantitative and qualitative data was executed as per standard statistical procedure and ANOVA such as standard error (SE), standard deviation (SD) and coefficient of variability (CV) etc. (Sukhatme and Amble, 1995).

The seed yield were collected as M1 generation for electrophoresis. The seed yield collected from all the treatments was used for the protein estimation and for electrophoresic assessment. The seeds were subjected for grinding to form the seed floor The 25 mg of seed powder was added to 1ml of Protein Extraction Buffer (0.2% SDS, 0.05 M Tris -HCL, 5 M Urea and 1% β-Mercaptethanol with pH adjusted as 6.8-7.00) and mixed thoroughly to extract the seed storage protein and subjected to the centrifugation(15000×g) for 7 Minutes at 4oC. Supernatant was collected for further study(Asghar et al., 2003). The protein estimation was carried out by means of Bradford assay(Bradford, 1976).

The 25 l protein extracts and 25l Laemmli buffer mixed together. The 50 µg seed protein from each treatment along parent control was mixed the sample buffer pH 6.8 (Laemmili Buffer) thereafter loaded in the gel wells. SDS- PAGE has been executed using

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11.25% polyacrylamide gel at 50 mA for two and half an hour. The 2 separate gels were run to check the reproducibility. The gel was stained with 0.2% (w/v) Coomassie Brilliant Blue R- 250 for 5 hours and then destained for 24 hours (Laemmli, 1970). The analysis of properly destained gels was executed with the help of gel documentation system. The molecular marker weight was loaded in the one of the gelwells alongwith seed protien of eachtreatments. The consistent bands present in the gel were taken into consideration and represnted in Figure9, Figure 10, and Figure 11. The gels were analyzed as (1) for presence of particular protein band and as (0) for absence of bands shown in Table 4. The Pair wise similarities was computed using Jaccard Similarity Coefficient between the control and its induced mutants. The cluster analysis has been executed on the basis of similarity matrix using UPGMA(Sneath and Sokal, 1973; Karihaloo et al, 2002).

. Jaccards Coefficient Sij = nij / ni + nj - nij

Where, Sijthe similarity between lanes i and j, nij the number of the similar corresponding bands for i and j, nitotal number of bands in present in the lane i, njtotal number of bands in present in the lane j, ni + nj - nij total number of bands present in both lane. The dissimilarity can be obtained by substracting similarity from one i.e. 1- Sij (Simlarity).

The clustering was performed using by UPGMA (Unweighted Pair Grouping of Mean of Arithmetic Average) Clustering Method –UPGMA (Arjen Van Ooyen, 2001).

RESULT AND DISCUSSION

The total normal and abnormal cells were scored to calculate the mitotic index for each treatment and presented in the table1. The mitotic index was observed as decreased or depressed as compared to the untreated control treatment. The maximum mitotic index was observed as 7.775 in the untreated control and minimum mitotic index was recorded as 4.28 in the combined treatments of SA and x-ray.In the present study all the treatment including independent and combined treatment has revealed the depressed mitotic index as compared to the control. The dose dependent increase mitotic aberration following chemical and physical mutagenic treatment has been reported in a fababean (Vandana and Dubey,1992a,b). The values of mitotic aberration in SA treatment has been reported as increasing in horsegram (Sirsat et al.,2010).The percent frequency of abnormal or aberrant cells has been reported as index of efficiency and effectiveness of mutagen (Kumar et al., 2003). The chemical mutagen sodium azide has been reported as more effective (Sirsat et al.,2010). The various mitotic abnormalities or irregularities observed in the each treatments is represented in the table 2.Stickiness- it is most common abnormality observed in all the treatment and depicted in table The frequency of this abnormality was more at metaphase. The abnormality might be attributed to the result due to cytochemically balanced reactions disturbances by the radiation effect (Jayabalan and Rao, 1987) while Tarar and Dnyansagar (1980a) reported it was due to depolymerization of nucleic acid caused by mutagenic treatment. The figure 3 (a) and (b)represent the mitotic abnormality stickiness. Polyploidy - It was observed more in all the treatment. The abnormality might be due to inactivness of spindle apparatus and failure of chromatid separation. The numbers of

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chemical have been reported as cytotoxic effect on spindle apparatus (Sharma and Sharma, 1990; Sudhakar et al., 2001). The figure 4 (a) and (b) represent the mitotic abnormality polyploidy. Ring formation – The ring formation was recorded in all the treatment. The clastogenic chromosomal aberration such as fragment, ring chromosome, which might be attributed to breakage and ring formation, might be due to the broken chromosome end (Kumar and Tripathi, 2003). The figure 5 (a) and (b) represent the mitotic abnormality ring formation. Chromatin bridge - The chromatin bridge was observed in all the treatment and the frequency is more in higher concentration of SA and x-ray. It might be attributed to the failure of chiasmata terminalization and chromosome would get stretched between poles (Saylor and Smith, 1966). The figure 6 (a) and (b) represent the mitotic abnormality chromatin bridge. Laggard - The frequency of laggard was more in some of the independent mutagenic treatment and combined treatment. The occurrence of lagging chromosome might be attributed to the failure to carry the chromosome to respective pole (Tarar and Dnyansagar, 1980b). The fragment might be due to broken chromosome fragment fail to reunite with the chromosome (Kaur and Grover, 1985). The figure7 (a) and (b) represent the mitotic abnormality lagging chromosomes. Micronucleus formation – The micronucleus was observed in the various treatments and mentioned in the table No.2. The micronucleus formation may be attributed to the failure of movement of lagging chromosome (Kumar and Dubey, 1998) fragment which fail to move toward the respective pole as well as the irregular distribution of acentric fragment (Bhattacharya, 1983). The figure 8 (a) and (b) represent the mitotic abnormality micronucleus formation in the wild chickpea. Mutagenic potentiality of combined treatment of SA and gamma rays has been reported as more efficient than their individual ones in cowpea (Kumar and Verma, 2011).Similar observation have been reported in wild chickpea (Kamble and Patil, 2014).

The total 20 bands in the T1 treatment were observed representing molecular weight range between 7.94 KDa to 119.08 Kda which is in the confirmity with the previous report(Kamble

and Patil, 2014),whereas the qualitative and quantitative variation were observed in all other treatments T2 to T10. The total number of bands observed in the untreated and induced mutants in the present study are T1=20, T2=22, T3=22, T4=21, T5=18, T6=17, T7=20, T8=21, T9=21 and T10=19. The total 18 and 22 bands has been reported in 21 accesion of kabuli chickpea (Cicer arietinum), showing range between 5 KDa to 70 KDa by using electrophoretic study(Asghar et al., 2003). The intense band was described as ‘major bands’ while less intense band as ‘minor band’.The range of major band was observed from 7 to 12. 12 major bands were observed in the control treatment while 7 to 11 was observed in the mutants while rests of the bands were minor band. The increase in the protein content has been reported in Phaseolus by using the mutagenic treatment and mutation breeding (Prasad et

al., 1986). There has been no any significant change in the seed protein content of mutant in the cultivated chickpea(Kharkwal, 1998 a, b; Wani and Anis, 2008). The polypeptide bands of different sizes ranging from 5.92 KDa to119.08 KDa were observed in all the treatments alongwith the untreated control. Pairwise similarity between parent and mutants has been derived on the basis of Jaccard’s coefficient ranged between 0.333 to 1.0 with a mean of 0.619 represented in Table 3. Figure 12 represents the dendrogram obtained by UPGMA

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(Unweighted Pair Group Method using Arithmetic Average Method) clustering of similarity matrix; similar observation has been mentioned in Solanum melanogena L. and its wild species (Karihaloo et al., 2002). Tallbery(1981 a, b) confirmed that the change in protein composition is owing to the mutated genes. Protein and their respective pattern with regards to appearance of new bands and disappearance of old band and relative mobility and colouration of band in mutants confirm alteration in polypeptides of seed protein due to gene mutation(Gottschalk and Wolff, 19983).

The protein profiling of the seed storage protein has been reported as one of the potential methods to differntiate the parents and mutants (Amjad et al., 2009). Jaccard Coefficient was computed on the basis of Unweighted Pair Group Method by Arithematic Mean (UPGMA). Some polypeptide band is present in its mutants while absent in parent control treatment. The mutants were polymorphic as compared to other mutants in M1 generation. Similar observation has been reported in Chrysanthemum (Kumar et al., 2006). Genetic distance between all 10 treatments varied from 0.333 to 0.75 as reveled by Jaccard similarity coefficient. Similar observation has been reported in Chrysanthemum and its radio-mutants(Kumar et al., 2006) in wild chickpea and its induced mutants treated with physical and chemical mutagens independntely and in combination (Kamble and Patil, 2014). The polymorphism was observed in the banding pattern in the present investigation.

The dendrogram derived with the help of Jaccard similarity coefficient by using UPGMA method which shows one control parent and its induced 9 mutant into two major clusters. The first cluster consists of T1 and seven induced mutants T5, T2, T3, T4, T8, T9 , T10, and the second cluster consists of mutants T6, T7. The second cluster shows protein diversity from parents and other mutants in M1 generation and depicted in the Figure 12. The SDS-PAGE Electrophoresis of seed storage proteins could be employed for the assessment of the genetic variation relative to germplasm and also to distinguish the mutants from their parent genotypes(Amajad et al.,2009).

3) The seed germination percentage has been observed decreased at higher doses. The inhibition of seed germination at higher doses of mutagen independently and in combination radiation might have resulted due damage to chromosome (Al- Safadi and Simon, 1996). However, T2, T3 and T5 treatments showed 80% germinability The delayed germination has been observed in all the treatments. Similar observation has been reported with increasing concentration of mutagenic treatment (Alka et al., 2007).The plant height were higher in T5, T7, T10 with maximum mean plant height 22.94 cm in T10

The maximum height induced in combination mutagenic treatments has been reported in chickpea (Wani and Anis, 2008). The significant higher plant height has been reported in grasspea (Waghmare and Mehra, 2000). No dose dependant relation has been observed. The number of primary branches was observed higher as compared to control and maximum mean 5.74 in T8 treatment and the number of secondary branches were also recorded as maximum in T8 treatment 5.14. The similar observation has been reported followed by the mutagenic treatment in grasspea (Waghmare and Mehra, 2000), in wild chickpea (Kamble and Petkar, 2015).The increase in number of flower except T7 and T10 has been observed in present study as compared to the control treatments. The number of pods was recorded maximum 8.83 in T8

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treatment. Wani and Anis (2008) have reported the significant quantitative increase in pod in chickpea induced by lower dose of mutagen. The higher number of pod per plants has been reported followed by mutagenic treatment as compared to control in grasspea (Waghmare and Mehra, 2000). The increase in variability for number of pods per plants following mutagenic treatment has been reported in khesari (Singh and Chaturvedi, 1990).The mutation inducing many traits could be attributed to the mutation of pleiotropic gene or mutation of gene cluster or chromosomal arrangement as has been reported in chickpea (Wani and Anis, 2008). The observations in present investigation revealed the conformity as reported in chickpea (Wani and Anis, 2008).

Table 1: Mitotic Index and Standard Deviation of the dividing cells in Mitotic cell division in wild Cicer reticulatum L.

Sr. No. Treatment Total Number of Dividing cells Mean (Normal + Abnormal)

Total cells scored Mean

Mitotic Index Mean± Standard Deviation (δ)

1 T1 90 1158 7.78(±0.1)2 T2 71 1232 5.76(±0.02)3 T3 72 1276 5.64(±0.02)4 T4 62 1128 5.49(±0.02)5 T5 64 1345 4.75(±0.06)6 T6 64 1422 4.50(±0.04)7 T7 61 1423 4.28(±0.05)8 T8 69 1015 6.79(±0.02)9 T9 67 1027 6.52(±0.01)10 T10 68 1068 6.36(±0.02)

Table 2: Various mitotic abnormalities and irregularities in wild Cicer reticulatum L.Sr. No.

Treatment

Stickiness

Polyploidy

Ring formation

Chromatin bridge

Laggard

Micronucleus formation

Total

1 T1 --- -- -- -- -- -- --2 T2 2 1 1 1 2 2 93 T3 2 2 1 1 3 2 114 T4 3 2 2 2 3 1 135 T5 3 3 2 2 2 1 136 T6 4 3 2 3 3 3 187 T7 4 3 3 3 3 3 198 T8 1 1 1 1 1 2 79 T9 1 1 1 1 1 2 710 T10 2 1 2 1 1 2 9

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Table 3: Similarity Matrix in M1 generation of wild chickpea and its induced mutants. (Cicer reticulatum L.).

T1 1

T2 0.68 1

T3 0.68 0.629 1

T4 0.64 0.592 0.72 1

T5 0.727 0.538 0.538 0.625 1

T6 0.48 0.392 0.392 0.407 0.521 1

T7 0.6 0.448 0.448 0.464 0.583 0.541 1

T8 0.576 0.482 0.535 0.615 0.5 0.407 0.518 1

T9 0.576 0.592 0.592 0.615 0.5 0.357 0.518 0.75 1

T10 0.625 0.413 0.464 0.481 0.48 0.333 0.444 0.538 0.538 1

6.584 5.086 4.689 4.207 3.584 2.638 2.48 2.288 1.538 1 34.094

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 55

Table 4: Polypeptide bands observed in control wild Chickpea and its induced mutants in M1 generation. (Cicer reticulatum L.).

Sr. No. Molecular Weight in KDa T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

1 119.08 1 1 1 1 1 1 1 1 1 12 110.22 0 0 0 0 0 1 1 1 0 03 109.12 1 0 0 0 1 0 0 0 0 14 108.92 0 1 0 0 0 0 0 0 0 05 107.53 0 0 0 1 0 0 0 0 0 06 96.56 1 1 1 1 1 1 1 0 0 17 85.32 0 1 0 0 0 0 1 0 1 08 84.91 0 0 1 1 0 0 0 0 0 09 83.97 1 0 0 0 1 1 1 1 0 1

10 81.73 0 0 0 0 0 0 0 1 0 011 73.01 1 1 1 1 1 0 1 1 1 112 68.47 0 0 0 0 0 1 0 0 0 013 65.75 1 1 1 1 1 0 1 1 1 014 60.19 0 0 0 0 0 1 0 0 0 015 59.21 0 0 0 0 0 0 1 0 0 016 58.18 1 1 1 1 0 1 1 1 1 117 56.45 0 0 0 0 0 0 0 0 0 118 54.31 0 0 0 0 0 0 0 1 1 019 52.39 1 1 1 1 1 1 1 1 1 020 44.77 1 1 1 1 1 1 1 1 1 121 39.31 0 0 0 0 1 1 1 0 0 022 38.26 1 1 1 1 1 1 1 1 1 123 36.96 0 0 0 1 0 0 0 1 1 1

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24 36.35 0 0 1 0 0 0 0 0 0 025 34.45 1 1 1 1 1 0 1 1 1 126 32.07 0 1 0 0 0 0 0 0 0 027 31.02 1 1 1 1 1 1 0 1 1 028 28.43 1 0 0 0 0 0 1 0 0 129 24.72 1 1 1 1 1 1 0 0 0 030 23.11 0 0 0 0 0 1 1 0 1 031 22.71 0 0 0 1 1 0 0 1 1 132 19.7 1 1 1 1 1 0 1 1 1 033 16.54 1 1 1 1 1 1 1 1 1 134 15.66 0 0 1 0 0 0 0 1 1 135 14.13 1 1 1 1 1 1 1 1 1 136 13.01 0 1 1 0 0 0 0 0 0 037 11.87 1 1 1 1 0 0 0 1 1 138 9.62 1 1 1 1 1 1 1 1 1 139 7.94 1 1 1 0 0 0 0 0 1 140 6.52 0 0 1 1 0 0 0 0 0 041 5.92 0 1 0 0 0 0 0 0 0 042 Total 20 22 22 21 18 17 20 21 21 19

Table 5: Effect of SA and radiation on Phenotypic characters in M1 Generation.Sr.

No

Treatment Days

Required

to

Germinate

Mean

(Days)

Germination

Percentage

Mean

Plant

Length

(in

cm)

30

DAS

Mean

Number

of

Primary

Branches

Mean

60 DAS

Mean

Length of

Primary

Branches

Mean (In cm)

Number

of

Secondary

Branches

Mean

70 DAS

Mean

Length of

Secondary

Branches

Mean (In

cm)

Number

Of

Flowers

100DAS

Mean

Number

of Pods

110 DAS

Mean

1 T1 . 3.3 100% 21.94 3.36 29.7 4.34 11.8 5.95 8.65

2 T2 7.5 80% 21.55 4.26 28.23 4.66 12.24 6.77 5.92

3 T3 7.9 80% 20.95 3.50 27.95 5.42 9.76 6.45 6.74

4 T4 8.5 70% 21.37 4.16 29.4 4.08 10.45 7.01 6.79

5 T5 9.3 80% 22.12 5.18 29.82 4.24 13.67 6.21 6.47

6 T6 7.2 70% 20.14 5.11 27.34 4.21 14.1 6.34 6.95

7 T7 9.6 60% 22.94 4.95 26.92 4.92 12.1 5.56 7.23

8 T8 9.4 70% 21.85 5.74 27.64 5.14 9.34 7.84 8.83

9 T9 9.5 70% 20.85 4.96 27.84 4.12 9.20 7.62 7.21

10 T10 8.7 60% 22.42 5.28 26.75 4.5 8.95 5.64 8.62

F-test Sig Sig Sig Sig Sig Sig Sig

SE(m±) 0.03 0.26 0.43 0.54 0.72 0.77 0.84

CD at 5% 0.12 0.34 0.59 0.76 1.48 1.27 2.44

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CONCLUSION

SA and X-ray reveal the mitotoxic and clastogenic effect in the present study. Both mutagenic agents showed the mitodepressive activity independently and in combination treatment. The potential and effectiveness of mutagenic treatment was found to be X-ray >S A> SA + X- ray in present study.

The chemical and physical mutagen showed the potential to cause the mutation in the wild chickpea. The SDS-PAGE electrophoretic protein profile in M1 generation represented the polymorphism in the banding pattern as compared to the control treatment. The variation was observed between control and its induced mutants. The SDS-PAGE electrophoretic pattern of the induced mutants represented the deviation from its untreated control parent with respect to the Jaccards similarity coefficient in the present Biochemical and Biotechnological assessment study. The second cluster shows more protein diversity from parents and other mutants.

The overall comparative study with respect to phenological parameter the T8 treatment appeared the fairly good treatment over all other treatments as it shows maximum qualitative traits and characteristics over all other treatments. ANOVA for all the treatments were observed significant for all phenotypic characters (p<0.05). The treatment with desirable character could be used in breeding programme. Similarly, ANOVA for genotypes were significant for all the characters t (p<0.05). The genotypes possessed desirable characters that could be directly produced after release and they could be used indirectly in breeding programme. The comparative result on overall variability was observed significant in present investigation.

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PHOTOPLATES

Figure 1. The Seeds of Cicer reticulatumL. Figure 2. T8 treatmentMitotic Chromosomal Abnormalities in wild chickpea treated with SA and X-rays.

Figure 3 (a) Stickiness Figure 3 (b). Stickiness Figure 4 (a). Polyploidy

Figure 4(b). Polyploidy Figure 5(a). Ring Formation

Figure 5(b). Ring Formation

Figure 6(a). Chromatin Bridge

Figure 6(b). Chromatin Bridge

Figure 7(a). Laggards

Figure 7(b). Laggards Figure8(a). Micronucleus Formation

Figure 8 (b). Micronucleus Formation

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Figure 9. Gel showing the banding pattern

Figure 10. Gel showing the banding pattern

Figure 11. Gel showing the banding pattern

Figure 12. Dendrogram based on Jaccards Similarity Coefficient of Seed Protein in Wild Control Parent and its induced Mutants by using UPGMA in M1 Generation