Assessment of mutagenicity induced by MMS and DES in Capsicum annuum L.

Abstract

Seeds of Capsicum annuum L. var. G4 were subjected to different concentrations of methyl methane sulphonate (MMS) and diethyl sulphate (DES). The effects of different mutagenic treatments on meiosis, chiasma frequency, and pollen fertility have been studied in M₁ generation. Various types of meiotic aberrations such as univalent, multivalent, stickiness, bridge, laggards, cytomixis etc. were observed in all the treatments. However, the MMS treatments proved to be more effective in inducing meiotic aberrations as compared to DES. Moreover, the frequency of meiotic aberrations was at its maximum at metaphase followed by anaphase and telophase stages. As the concentrations increase, reduction in chiasma frequency and pollen fertility was observed in all the treatments and, MMS again was found to be more effective than DES treatments.

1. Introduction

Induced mutation has great relevance in raising superior plant types in different crop plants. Most of the mutations are lethal or semi-lethal and do not have any practical value possibly either due to doses monitored or mutagens employed. Selection of efficient mutagens and their treatment doses is prerequisite for successful mutagenesis as mutagens are potential tools for direct improvement of certain qualitative and quantitative characters. Mutagenesis is usually done to create genetic variability not available in the gene pool or to repair specific deficiency of an otherwise outstanding genotype. Genetic variation can be artificially induced by the use of physical and chemical mutagens as they affect a wide range of chromosomal alterations resulting into abnormal behavior during mitosis and meiosis leading to various degrees of sterility and inducing various structural changes in the chromosomes. The most common form of sterility is the occurrence of non-functional gametes. Mutation induced chromosomal anomalies are the primary basis of genetic changes. Thus, cytogenetical investigation is an important source of information regarding the genetical hazards due to mutagens as they deal with the genetic material, the chromosomes, and more appropriately the DNA which controls the phenotype. It also provides a considerable clue to assess the sensitivity of plants for different mutagens and to ascertain the most effective mutagens and their treatment doses for a given crop to realize maximum results.

Capsicum annuum L. (2n = 24), commonly known as chili or pepper, is a spice yielding and a cash crop of India as well as other countries the worldover. Its fruits are used to stimulate gastric activities and to increase blood circulation. It is also a stimulant and carminative used for neuralgia and rheumatism. In the present investigation, attempts have been made to explore the possibilities of inducing alterations in the genotype to enhance the genetic variability through the use of chemical mutagens i.e., methyl methane sulphonate (MMS) and diethyl sulphate (DES) in C. annuum L. var. G4 in the M₁ generation.

2. Materials and methods

Healthy, pure and dry seeds (moisture content 10–12%) of C. annuum L. var. G4 obtained from Indian Agriculture Research Institute, New Delhi, were used. For mutagenic treatments of MMS and DES, seeds were first presoaked in distilled water for 12 h and then treated with different concentrations i.e. (0.01%, 0.02%, 0.03%, 0.04%, and 0.05%) of freshly prepared aqueous solution of MMS and DES in phosphate buffer (pH = 7.0) for 6 h. After the treatment period, the treated seeds were thoroughly washed in running tap water to remove the residual effects of mutagens, if any. The treated seeds along with the untreated (control) were sown in earthen pots to raise M₁ generation. After 30 days the seedlings were transplanted in the field at the Faculty of agricultural Sciences, AMU, Aligarh. For meiotic studies young flower buds from 30–40 randomly selected M₁ plants were kept in a freshly prepared Carnoy’s solution for 24 h, washed and than preserved in 70% alcohol at 4 °C. Squashing was done in 2% acetocarmine and the slides were made permanent by dehydration in buytl-alcohol series followed by mounting on Canada balsam. Pollen sterility was assessed by staining pollen in 2% acetocarmine where, stained pollen grains with regular outline were considered as fertile.

3. Results

Normal meiosis with 12 bivalents at diakinesis (Fig. 1a) was observed in control plants. In the treated plants, however, different kinds and frequencies of meiotic chromosomal aberrations at different divisional phases were encountered during microsporogenesis. The most frequent aberrations were multivalents, stickiness, precocious separation, and stray bivalents at metaphase I/II. Laggards, bridges, and unequal separation at anaphase I/II, and disturbed polarity, micronuclei, and cytomixis were seen at telophase I/II. Bridges at telophase I/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(a–i). 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 dose-dependent increase in the meiotic aberrations was observed in all the mutagenic treatments. The maximum frequency of aberration was at highest concentrations of both the mutagens and was more in MMS treatments than DES. A dose-dependent reduction in pollen fertility was recorded in plants treated with mutagens and it was positively correlated with the meiotic aberrations. The MMS treatments showed more reduction in pollen fertility as compared to the DES treatments (Table 2). A dose dependent decrease in chiasma frequency per cell and per bivalent was recorded in treated plants. However, reduction in chiasma frequency was higher in MMS treatments than DES (Table 3).

Figure 1.

Representative microphotograph of PMC with meiotic aberrations induced by MMS and DES in Capsicum annuum L. (a) Twelve bivalents at diakinesis (control); (b) precocious separation of chromosome at metaphase; (c) stray bivalents at metaphase-I; (d) Laggards at anaphase-I; (e) Laggards at anaphase-II; (f) sticky bridge at telophase-II; (g) disturbed polarity and micronuclei at telophase-II; (h) multinuclei at telophase-II; (i) migration of chromosomes from one cell to another (cytomixis).

Table 1.

Abnormalities (%) at different stages of meiosis induced by (MMS) and (DES) in Capsicum annuum L. var. G4.

Conc.	Total PMC’s observed	Total abnormal PMC’s observed	Metaphase I/II					Anaphase I/II			Telophase I/II				Total aberration (%)
			Uni.	Multi.	Stic.	Preco.	Stray.	Lag.	Bri.	Uneq.	Micr.	Dist.	Cyto	Bri.
Control	200	–	–	–	–	–	–	–	–	–	–	–	–	–	–
MMS (%)
0.01	212	16	–	(2)0.94	(2)0.94	(1)0.47	(3)1.41	(2)0.94	–	(1)0.47	–	(3)1.41	(2)0.94	–	7.55
0.02	208	23	(1)0.48	(3)1.44	(4)0.92	(2)0.96	(1)0.48	(1)0.48	–	(2)0.96	(1)0.48	(3)1.44	(4)1.92	(1)0.48	11.06
0.03	215	30	(2)0.93	(4)1.86	(4)1.86	(1)0.46	(2)0.93	(3)1.39	(2)0.93	(1)0.47	(2)0.93	(3)1.39	(4)1.86	(2)0.93	13.96
0.04	220	42	(3)1.36	(4)1.86	(5)2.27	(2)0.90	(3)1.36	(4)1.81	(3)1.36	(4)1.81	(5)2.27	(4)1.81	(3)1.36	(2)0.92	19.09
0.05	215	58	(4)1.86	(5)2.32	(6)2.79	(3)1.39	(4)1.86	(5)2.32	(4)1.86	(5)2.32	(6)2.79	(7)3.25	(5)2.32	(4)1.86	26.97
DES (%)
0.01	207	10	–	(1)0.48	(2)0.96	–	(1)0.48	(1)0.48	–	–	(2)0.96	(2)0.96	–	–	4.84
0.02	210	16	(1)0.47	(2)0.95	(2)0.95	(1)0.47	(2)0.95	(2)0.95	–	(1)0.47	(2)0.95	(2)0.95	(1)0.47	–	7.62
0.03	215	24	(2)0.93	(3)1.39	(3)1.39	(1)0.46	(2)0.96	(3)1.39	(1)0.46	(2)0.93	(3)1.39	(3)1.39	(1)0.46	–	11.17
0.04	220	37	(3)1.36	(4)1.81	(4)2.27	(2)0.90	(3)1.36	(4)1.82	(2)0.90	(4)1.81	(4)1.81	(4)1.81	(2)0.90	(1)0.45	16.82
0.05	225	51	(4)1.77	(5)2.22	(6)2.66	(3)1.33	(4)1.77	(5)2.22	(3)1.34	(5)2.23	(6)2.67	(5)2.22	(3)1.34	(2)0.88	22.66

Conc. = concentration, Uni. = univalent, Multi. = multivalent, Stic. = stickiness, Preco. = precocious separation, Stray. = stray bivalent, Lag. = laggard, Bri. = bridge, Uneq. = unequal separation, Micro. = micronuclei, Dist. = disturbed polarity, Cyto. = cytomixis. Within parenthesis is the No. of abnormal cells. – = not observed.

Table 2.

Effect of MMS and DES on pollen fertility in M1 generation of Capsicum annuum L. var. G4.

Mutagens	Concentration (%)	Pollen fertility (%)	Reduction (%)
Control	–	94.80	–
MMS	0.01	84.54	10.82
	0.02	80.10	15.50
	0.03	73.25	22.73
	0.04	66.55	29.79
	0.05	59.85	36.86
DES	0.01	88.15	7.01
	0.02	82.60	12.86
	0.03	77.90	17.82
	0.04	69.70	26.47
	0.05	63.35	33.17

Table 3.

Effect of MMS and DES on chiasma frequency at metaphase-1 in M1 generation of Capsicum annuum L. var. G4.

Treatments	No. of chaisma per cell	No. of chiasma per bivalent
Control	18.77	1.56
MMS (%)
0.01	18.10	1.50
0.02	17.30	1.44
0.03	16.80	1.40
0.04	15.10	1.25
0.05	14.20	1.18
DES (%)
0.01	18.40	1.53
0.02	17.65	1.47
0.03	17.10	1.42
0.04	15.25	1.27
0.05	14.40	1.20

4. Discussion

In the present investigation similar types of meiotic abnormalities were found in all mutagenic treatments but the frequency of abnormalities was different in different treatments, indicating the different mutagenic potentials of mutagens against C. annuum L. Bivalents were found clumped in single or different groups at metaphase I due to stickiness. Jayabalan and Rao (1987) suggested that stickiness of chromosomes might be due to disturbances in cytochemically balanced reactions in the nucleic acids. However, it seems most probable that some kinds of gene mutations lead to incorrect coding of some non-histone proteins involved in chromosome organization and leads to chromosomes clumping (Gulfishan et al., 2010). It may also be possible that the mutagen itself reacts with the histone proteins and brings about a change in the surface property of chromosomes due to an improper folding of DNA, thereby causing them to clump or stick together (Gaulden, 1987). The occurrence of univalents and multivalents at metaphase-I has been reported in various plants like barley (Kumar and Singh, 2003) and broad beans (Bhat et al., 2005). Mutagen induced structural changes in chromosomes might be responsible for the failure of pairing among homologous chromosomes and hence the occurrence of univalents. The mutagen induced translocations and possibly inversion might be involved in the formation of multivalents. Precocious chromosome migration to the poles may have resulted from spindle dysfunction or precocious chiasma terminalization at diakinesis or metaphase-I (Kumar and Rai, 2007). Stray bivalents at metaphase-I seem to be caused by spindle dysfunction and clumping of chromosomes (Bhat et al., 2007). The presence of laggards may be attributed to the inability of multivalents to separate properly (Ganai et al., 2005). Laggards may be explained on the basis of abnormal spindle formation and chromosomal breakage. According to Tarar and Dnyansagar (1980), unsynchronized bivalents or laggards might be due to the discrepancies in spindle formation. Laggard at anaphase can be attributed to the delayed terminalization or perhaps to stickiness of chromosomal ends (Minija et al., 1999). The formation of chromatin bridges might be due the failure of chiasmata in a bivalent to terminalize and the chromosome gets stretched between the poles (Saylor and Smith, 1966), whereas, Bhattacharjee, 1953) bridge formation was attributed to the interlocking of bivalent chromosomes. Unequal separation of chromosomes at anaphase I/II, observed in the present study, may be due to the occurrence of multivalents and failure of chromosomes to segregate equally. Micronuclei as observed in the present study at telophase I/II may be due to the association of fragments and lagging chromosomes which failed to reach the poles and got included in the daughter nuclei (Kumar and Dubey, 1980). Cytomixis generally refers to the migration of chromatin from one cell to the other through cytoplasmic connections. The transmigration of chromatin material with cytomictic connections might have resulted in altered numbers of chromosomes. Variation in chromosome number in few pollen mother cells may be due to cytomixis, which is considered as a source of production of aneuploid and polyploid gametes (Koul, 1990; Yeng et al., 1993); Chiasma frequency was variable in the populations treated with different concentrations of the mutagens, however, reduction in chiasma frequency was dose dependent. A considerable decrease in chiasma frequency showed the prominent effect of MMS and DES on chromosomes. Rees (1955) showed that crossing over and chiasma formation are under genetic control. Goud (1967) in wheat, Sree Ramulu (1973) in Sorghum and Sadanandam and Subhash, 1984) in Capsicum have also reported the reduction in chiasma frequency due to mutagen induced structural changes. In the present investigation reduction in chiasma frequency may be attributed to the nature and potency of mutagen and to the underlying factors such as complex structural changes or to the nature of the genes responsible for chiasma formation. Pollen fertility is the index of meiotic behavior. Greater the chromosomal aberration greater will be the pollen sterility. This is in agreement with many workers (Nerker, 1970; Reddy and Annadurai, 1991; Bhamburker and Bhalla, 1985) who have also reported dose dependent decrease in pollen fertility. Reduction in pollen fertility observed in treated population is attributed to the vast array of meiotic aberrations that were induced by mutagens leading to the formation of aberrant pollen grains (Rana and Swaminathan, 1964; Sinha and Godward, 1972). The reasons of pollen sterility may also be due to gene mutation or invisible deficiencies.

The cytogenetic abnormalities have been regarded as one dependable parameter for estimating the mutagenic potential of a mutagen and it can be judged by the percentage of abnormalities it induces. The genetic changes brought about by the mutagens provide good scope for further improvement of this crop. The present study, thus, envisages the use of MMS as compared to DES, in various concentrations to induce mutations and the positive mutation in economic characters may be selected and tested for the improvement of C. annuum L. var.G4.