International Journal of Botany and Research (IJBR) ISSN 2277-4815 Vol. 3, Issue 2, Jun 2013, 79-86 TJPRC Pvt.

. Ltd.

GENOMIC DAMAGE INDUCED BY INDIVIDUAL AND COMBINATION TREATMENT OF GAMMA RAYS AND ETHYL METHANE SULPHONATE IN CORIANDRUM SATIVUM L VAR. KARISHMA
IRAM FATMA JAFRI, AINUL HAQ KHAN & MOHD GULFISHAN Cytogenetics and Mutation Breeding Laboratory, Department of Botany, A.M.U. Aligarh, India

ABSTRACT
Aim of the present study is to find out the mutagenic effects of individual and combination treatment of gamma rays and EMS on Corriandrum sativum L. Healthy cremocarp were subjected to different doses of gamma irradiation and EMS in individual and combination set. During microsporogenesis, meiotic investigation of young floral buds was carried out in treated as well as un-treated plant materials. Meiotic study clearly exposed the meiotic malfunctioning of pollen mother cells (PMCs) that had shared various types of cytological abnormalities such as univalents, multivalents, stickiness, precocious movement, stray bivalent, non-orientation, cytomixis, laggard, bridges, unequal separation, micronuclei and disturbed polarities. However, this impairing during meiosis has found to be collinearly associated with concentration i.e. inclining tendency of abnormality percentage along with increasing dose/concentration of mutagens were registered. Perhaps aforementioned chromosomal aberrations may be introduced by asymmetrical distribution of chromatin material in PMCs, had definitely compromised with pollen fertility, resulting the increased frequency of pollen sterility.

KEYWORDS: Gamma Rays, EMS, Corriandrum sativum L, Chromosomal Aberrations INTRODUCTION

Coriander (Coriandrum sativum L.) is an important spice and aromatic herb that belongs to the family umbelliferae/Apiaceae (Hedburg and Hedburg, 2003). It is used in culinary (Diederichsen, 1996), medicine (Kubo et al., 2004; Delaquis et al., 2002) and; its green foliage rich in vitamins and other minerals is used in vegetables and salads. Seeds can be used as a spice and contain essential oils rich in linalool (Singh et al., 2005).because of its importance there is need to improve coriander variety to get better yield. Mutation breeding has become an important tool for the improvement of many crops through the mutation and modification in gene structure. Induced mutagenesis is a significant tool to break through the limitations of variability and to create variability in a short period of time. The degree of cytological aberrations in either mitosis or meiosis is regarded as one of the dependable criteria for estimating the potency of mutagen and to elucidate the response of various genotypes to a particular mutagen (Kumar and Sing 2003). The present study was undertaken to assess the effectiveness of physical and chemical mutagen on coriander veriety. Vary little cytogenetic work have been done in case of coriander. Patil and Bhatia (1992) reported that the localization of breaks along the chromosomes result from the affinity of EMS for guanine rich areas on the other hand the radiations can have direct effect on chromosomes. They may directly break chromosomes or alter one of the DNA bases or indirectly may initiate a chain of chemical reactions (Gupta (1989). Studies suggested that gamma rays and EMS are effective mutagens and can be used in inducing genetic variability in a number of crop plants (kumar and singh 2003). Keeping all these points in view, an experiment has been conducted to find out the comparative mutagenic effects of gamma rays, EMS and their combination treatment in Coriandrum sativum L. variety karishma.

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MATERIALS AND METHODS

Healthy and dry seeds of coriander variety karishma were used in the present investigation; this variety is well adapted to agro-climatic conditions of Uttar Pradesh. Homogeneous cremocarp were irradiated with 4 doses of gamma rays viz.10kR at 20kR, 30kR, and 40 kR from CO60 source at NBRI, Lucknow. In second set the seeds presoaked in distilled water for 12 hours at room temperature (251 oC) and then treated with different concentrations of mutagen (0.1, 0.2, 0.3 and 0.4%) for 6 hours. In another set seeds were also subjected to combined doses of gamma rays and EMS. During the mutagenic treatment frequent shaking was given throughout the treatment period to facilitate sufficient aeration. Seeds of control set were left untreated. After the treatment was over, the seeds were thoroughly washed in running tap water to remove the residual effect of mutagen sticking to the seed coat. Treated as well as untreated (control) seeds were sown in 5 replicates in a complete randomized block design (CRBD) (of 2 x 1 m2 each.) in Rabi season in the field at Department of Agriculture, Aligarh Muslim University Aligarh. The distance between the seeds along the row was kept at 10 cm whereas row to row distance was maintained at 20 cm. in each experimental plot. The young flower buds of proper size were collected carefully from treated as well as control populations and fixed in freshly prepared Carnoys fixative (absolute alcohol : chloroform : acetic acid, 6:3:1 ratio ) with a few crystals of ferric chloride (FeCl 3) for 24 hours. Thereafter, the materials were washed and preserved in 70% alcohol. Anthers were squashed in 1% propionocarmine.

EXPERIMENTAL RESULTS
Meiosis was normal in the control plants and showed regular formation of eleven perfect bivalents (2n = 22) at diakinesis(fig.1a) and metaphase I (fig.1b) fallowed by normal separation (11:11) at anaphase I (fig.1c). However, various chromosomal abnormalities were recorded in the plants raised from gamma rays, EMS and combination treatment. The most frequent aberrations were univalent, multivalents, stickiness (Fig.1, d), stray bivalents(Fig.1, e) and precocious separation (Fig.1, f) at metaphase I/II. Laggards (Fig.1, g) bridges (Fig.1, h,j) and unequal separation(Fig.1, i) at anaphase I/II, and disturbed polarity, micronuclei, (Fig.1, k) and cytomixis(Fig.1,l) were seen at telophase I/II. Bridges at telophaseI/II were also noticed in some pollen mother cells (PMCs) but in a very low frequency. The representative cytological features are shown in Fig. 1(ai). The frequency and spectrum of various meiotic aberrations in each treatment of both the mutagens along with the total percentage of abnormal cells have been summarized in Table 1. A dosedependent increase in the meiotic aberrations was observed in all the mutagenic treatments. The maximum frequency of aberration was at highest concentrations of each mutagen and was more in combination treatment then individual. A dosedependent reduction in pollen fertility was recorded in plants treated with mutagens and it was positively correlated with the meiotic aberrations graph1. The combination treatments showed more reduction in pollen fertility as compared to the individual treatments (Table 2). From the aforesaid result it becomes clear that the EMS treatment elicited a better response in inducing the chromosomal aberrations than gamma rays. It was found that various parameters under study were not equally affected by any one of the two mutagens, indicating the different mutagenic potentials of mutagens against Corianderum sativum L.

DISCUSSIONS
Physical and chemical mutagens are known to produce chromosomal aberrations leading to abnormal chromosome behavior during meiosis and consequently giving rise varying degree of sterility. In the present investigation a vast array of meiotic aberrations were recorded in the plants raised from the seeds treated with different doses/concentrations of EMS, gamma-rays and their combination treatments in coriander variety karishma. Mutagens have remarkable possibilities of causing variations in various qualitative and quantitative characters of plants by altering the

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genetic architecture. The chemical mutagens have been reported to be more potent in inducing mutations than the physical ones (Sharma 1965). Pollen fertility is an index of meiotic behavior of chromosomes. In the present investigation, pollen fertility decreased with the increase in concentrations of both the mutagens in coriander (var. karishma). The chromosomal behavior during meiosis is considered to be one of the most reliable indices for estimating the potency of mutagens and the response of a genotype to mutation. Mutagen induced structural changes in chromosomes and gene mutations might be responsible for the failure of pairing among homologous chromosomes and hence the presence of univalents. According to Katiyar (1978) alterations in chromosomal associations, composed of uni-, tri-, tetra-, and multivalent were possibly the outcome of non or irregular pairing of chromosomes due to translocation. Stickiness could be due to depolymerisation of nucleic acid caused by mutagenic treatments or due to partial dissociation of the nucleoproteinsand alteration in their pattern of organization (Evans, 1962, Jafri et. al, 2011). Jayabalan and Rao (1987) suggested that stickiness might be due to disturbances in the cytochemically balanced reaction. Precocious movement of chromosome as observed during the present investigation, probably caused by spindle disfunction (Gulfishan et al., 2010). Non-orientation and scattering of

chromosomes at metaphase I was observed in the present investigation which may be due to either the inhibition of spindle formation or the destruction of spindle fibers (Kumar and Rai, 2007). Non-orientation of chromosomes at metaphase I sometimes leads to unequal separation of chromosomes (10:12) as observed in the present investigation, may be due failure of chromosomes to reach to their poles. Sinha and Godward (1972) attributed unequal distribution to the occurrence of multivalents and failure of chromosomes to segregate equally. According to Kumar and Singh (2003) random movement of univalents to anyone of the poles leads to the unequal separation of chromosomes. Non-synchronous movement may be due to severe disturbance in spindle mechanism (Minija et al., 1999). The laggards observed during the present study might be due to delayed terminalisation, stickiness of chromosomal ends or because of failure of chromosomal movement. According to Bhattacharjee (1953), acentric

fragments or laggards may result in the formation of micronuclei at telophase-II and ultimately variation in number and size of pollen grains resulting from a mother cell. Saylar and Smith (1966) suggested that the formation of chromatin bridges might be due to the failure of chiasmata in bivalent to terminate and chromosome get stretched between the poles. In the present study, bridge formation may be attributed to the general stickiness of chromosomes at metaphase stage or breakage and reunion of chromosomes. Micronuclei might have arisen from the fragments and lagging chromosomes which failed to reach to the poles and get included in the daughter nuclei (Kumar and Dubey, 1998). Disturbed Polarity at anaphase and telophase stages seen in the present case may be due to spindle disturbances. Chromatin transfer from cell to cell or the connection between cells through cytoplasmic channels revealed the occurrence of cytomixis, these channels probably originated in the form of small projections that by elongating would touch another cell. The cell wall may dissolve at the point of contact forming a passage for chromatin or chromosome transfer (Maria et al., 1997). According to Sheidai et al. (2006) the plasmodesmata become completely obstructed by the deposition of callose but in some cases, they still persist during meiosis and increase in size, forming conspicuous inter-meiocyte connection or cytomictic channels that permit the transfer of chromosomes. From the aforesaid result it becomes clear that combination treatment elicited a better response in inducing the chromosomal aberrations than individual one. The relationship between aberrations and sterility suggested that mutagen induced sterility was mainly the result of chromosomal aberrations. The results also showed co-linearity between the concentration of mutagen and the percentage of chromosomal anomalies.

ACKNOWLEDGEMENTS
The authors are thankful to the University Grants Commission (UGC), New Delhi, India for financial assistance

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in the form of Non NET-UGC fellowship and to the Head Floriculture Section NBRI Lucknow, for irradiation of the seeds.

REFERENCES
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10. Jayabalan N , Rao G R (1987). Gamma radiation induced cytological abnormalities in Lycopersicon esculentum Mill. Var. Pusa Ruby. Cytologia 52: 1-4. 11. Katiyar R B (1978). Radiocytogenetical studies on Capsicum 1 Meiotic anomalies. Cytologia 43: 415-421 12. Khan A H, Sharma M, Jafri I F (2010). Clastogenci Effects of Single And Combined Treatments of MMS And Gamma Rays In Vicia faba L. Nucleus 52:145-152 13. Kimball R F (1997). The mutagenicity of hydrazine and some of its derivatives.Mut.Res. 39: 111-126. 14. Kleinhofs A Owais W M, Nilan RA (1978). Azide Mut Res 55: 165-195. 15. Kubo I., Fujita K., Kubo A., Nihei, K. and Ogura, T. 2004. Antibacterial activity of coriander volatile compounds against Salmonella choleraesuis. Journal of Agriculture and Food Chemistry 52(11):3329-3332 16. Kumar G, Rai P K (2007). EMS induced karyomorphological variations in maize ( Zea mays L.) Inbreds. Turk Biol 31: 187-195 17. Kumar G, Singh V (2003). Comparative analysis of meiotic abnormalities induced by gamma rays and EMS in barley. J Indian Bot Soc 82: 19-22. 18. Kumar S, Dubey D K (1998). Effect of separate and simultaneous application of gamma rays and EMS on germination, growth, fertility and yield in cultivars Nirmal and LSD-3 of khesari (Lathyrus sativus L.). J Phytol Res 11: 165-170.

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19. Maria de Souza A, Pagliarini M S (1997). Cytomixis in Brassica napus var. Oleifera and Brassica compestris var. Oleifera (Brassicaceae). Cytologia 62: 25-29. 20. Minija J Tazo A , Thoppil J E (1999). Mitoclastic properties of MentEhe rotudifolia L. J Cytol Genetics 34:169171. 21. Patil B , Bhat G I (1992). A comparative study of MH and EMS in the induction of chromosomal aberrations on lasteral root meristem in Clitoria ternatea L. Cytologia 57:259-264 22. Saylor L G, Smith B N (1966). Meiotic irregularities in species of inter-specific hybrids in Pisum Am J Bot 53: 453-468. 23. Sharma B (1965). Comparative study of physical and chemical mutagens on the basis of variations and appearing in second generation. Izvstia timiriazev agric acad Moscow USSR 4: 127-140. 24. Sheidai M, Attaei S, Masoomeh K P (2006). Cytology of some Iranian Stipa (poaceae) species and populations. Acta Bot croat 65: 1-11. 25. Sinha S S N, Godward M B E (1972). Radiation studies in Lens culinaris Indian J Genet 32: 331-339.

APPENDICES

Figure 1: (a-l), Representative Meiotic Features Observed in Control and Mutagen Treated Plants of Criandrum Sativum L. a) PMC Showing 11 Bivalents at Diakinesis, b) PMC Showing Normal Metaphase-I c) PMC Showing Normal Anaphase-I with 11: 11 Segregation, d) PMC Showing Stickiness at Metaphase-I e) PMC Showing Stray Bivalent at Metaphase-I f), PMC Showing Precocious Separation at Metaphase-I, g) PMC Showing Laggard at Anaphase I, h) PMC Showing Bridge at Anaphase-I, i) PMC Showing Unequal Separation(12:10) at Anaphase-I, j) PMC Showing Chromatin Bridge at Anaphase-II, k) PMC Showing Micronuclei Telophase -II, l) PMCs Showing Chromosome Transmigration Through a Cytoplasmic Channel.

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Table 1: Frequency of Meiotic Aberrations Induced by EMS, Gamma-Rays and Their Combination Treatments in M1 Generation of Coriandrum sativum L.) Var. Karishma
Total no. Of pmc Metaphase I/II Multivalent Precocious Separation Disturbed Metaphase Stickiness Univalent Stray Bivalent Anaphase I/II Micronuclei Unequal Seperation Lagard Bridge Telophase I/II Total % of Abnomalities 4.18 8.76 13.07 19.05 4.12 7.98 11.36 16.68 7.62 11.48 19.20 23.59 Cytomixis 0.36 0.77 1.30 0.41 0.76 1.22 1.59 0.39 0.82 1.20 1.29 Disturbed Polarity 0.76 1.10 1.54 2.17 0.41 0.76 0.81 1.19 0.78 1.23 1.60 2.56

Control 0.10% EMS 0.20% 0.30% 0.40% 10kR

Treatment

280 260 271 259 230 240 263 244 251 258 243 249 232

0.38 0.73 1.15 1.73 0.38 0.40 0.79 0.39 0.41 1.20 2.15

0.38 1.10 1.54 1.73 0.76 1.22 1.59 0.78 1.23 2.00 2.56

0.76 1.10 1.15 2.17 0.41 0.76 1.22 1.99 0.78 1.23 2.40 2.15

0.36 0.77 0.86 0.83 1.14 1.63 1.99 0.39 1.23 1.60 1.29

0.38 0.73 1.15 1.73 0.41 0.76 0.81 1.19 0.78 0.82 1.60 2.15

0.36 0.77 1.30 0.40 0.79 0.39 0.41 1.20 1.72

0.38 0.73 1.15 1.73 0.41 0.76 1.22 1.59 0.78 0.82 2.00 2.15

0.76 1.10 1.54 2.17 0.83 1.14 1.22 1.59 1.17 1.64 2.00 2.56

0.36 0.77 1.30 0.41 0.38 0.81 1.59 0.78 0.82 1.20 1.72

0.38 0.73 0.77 0.86 0.38 0.40 0.79 0.39 0.82 1.20 1.29

g -rays g -rays+ EMS

20kR 30kR 40kR 20kR+ 0.1% 20kR+ 0.2% 20kR+ 0.3% 20kR+ 0.4%

Table 2: Effect of EMS, Gamma -Rays and Their Combination Treatments on Pollen Fertility in M1 Generation of Coriandrum sativum L. Var. Karishma

Treatment Contro 0.10% 0.20% 0.30% 0.40% 10kR 20kR 30kR 40kR 20kR+0.1% 20kR+0.2% 20kR+0.3% 20kR+0.4%

Pollen Fertility (%) 96.29 87.09 76.54 69.69 62.74 88.88 79.16 71.87 65.07 81.63 73.64 64.51 55.96

Reduction (%) 9.55 20.51 27.62 34.84 7.67 17.79 25.36 32.42 15.22 23.52 33 41.88

g-rays + EMS

g-rays

EMS

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