Nigella sativa L

International Journal on Agricultural Sciences 8 (1) : 108-118, January-June 2017 ISSN No.: 0976-450X

RESEARCH PAPER

Chemical Mutagen affects Pollination and Locule Formation in Capsules of Black Cumin (Nigella sativa L.) AADIL YOUSUF TANTRAY*, AAMIR RAINA, SHAHNAWAZ KHURSHEED, RUHUL AMIN, SAMIULLAH KHAN Department of Botany, Aligarh Muslim University, Aligarh, Uttar Pradesh, India Received: 27 Febraury 2017

Revision: 20 March 2017

Accepted: 15 April 2017

ABSTRACT In the present study, comparative mutagenicity of an alkylating agent (EMS) alone and in combination with biodissociating agent (DMSO) was investigated in Nigella sativa L. The cytogenetic effect was scrutinized on division of pollen mother cell in Nigella sativa L. with the treatment of EMS alone and in combination with DMSO. The increase in mutagenicity was concentration- and duration-dependent in EMS and EMS+DMSO. The combination treatment resulted in the lowest significant negative deviation in cytometric differentia. The EMS treatment showed large deviation of mutagenicity in comparison to EMS+DMSO treatment. The validation of this experiment has been achieved by contrasting the different physio-morphological multi-genic differentia. Phenotypic expression was assessed by the quantitative and qualitative analysis of physio-morphological characters, manifested considerable deviations in all the treatments. The positive significant deviation was observed in 1% EMS + 2% DMSO treatment for 6 h when compared with the control. Interestingly, 2% EMS treatment for 9 h resulted in highest negative significant deviation. The population of 1% EMS + 2% DMSO treatment for 6 h had shown significant increase in total leaf area of plant (20.31%), with the increase in number of leaflets. The pollination mechanism has been changed from open-pollination into the complete selfpollination, which promotes the seed set in this crop by 16.02% when compared with control. Also, the number of locules per capsule was increased to 12 in 1%EMS+2%DMSO treatment given for 6 h, when compared with the control (5-6 locules/capsule). Key words: Nigella sativa, ethyl methyl sulfonate (EMS), di-methyl sulfoxide (DMSO), cytometric analysis and physio-morphological traits.

INTRODUCTION Nigella sativa L. (Black Cumin) is a spice yielding annual herb, with huge commercial and medicinal significance. It is used largely in traditional system of medicine for curing diabetes, asthma, bronchitis, headache, paralysis, hemiplegia, infection, inflammation and in gastro intestinal problems such as dyspepsia, diarrhea, dysentery and flatulence (Haddad et al., 2003). Datta et al., (2012) reported that this plant is cultivated across India, especially in Punjab, Bihar and Bengal. The species is also grown in many parts of world such as Lebanon, Israel and South Europe. Most evidences showed that black cumin is native of Middle East and Western Asia (Iqbal et al., 2010). This species Corresponding author: [email protected]

108

thrives well in cool-dry to warm-humid areas with sandy loam soil rich in microbial activity. The most suitable soil pH for cultivation is 7.0 to 7.5. There is the need of increase the seed set in this crop, as the seed contain important metabolite-thymoquinone, which is anti-cancer. Due to multi-ovary condition and less anthesis period of the plant, cross-pollination is not efficient to fertilize the whole ovules in this crop. So, complete self-pollination is the only way to ensure the maximum ovule fertilization at the time of anthesis. Mutation breeding is generally used to improve traits in crops as high seed yield, oil content, sweeter fruits etc. To enhance variability for crop improvement, mutagenesis is a potent method. Different mutants of

International Journal on Agricultural Science (IJAS)

AADIL YOUSUF TANTRAY et al.,

N. sativa have been developed earlier such as bushy, feathery leaf, lax branching, early flowering, dwarf, brown seed coat for different quantitative traits (Datta et al., 1986). Chemical mutagenesis is regarded as an effective and coherent tool in improving the yield and economically important traits of crop plants. The genetic makeup of economic crops has been improved by mutation breeding along with significant increase in crop production (Adamu et al., 2007). An alkylating agent, EMS induces miss pairing and base change due to chemical modification of nucleotides (Greene et al., 2003). It also induces chromosomal aberrations & variations in bio-chemical content (Mendhulkar et al., 2015). DMSO is not a mutagen itself but act as a carrier by dissolving non-polar and polar compounds of the living material. Morphological mutations also play a vital role in mutation breeding programs. A wide range of mutant phenotypes were induced in the present experiment, several of which are useful from the breeder's point of view. N. sativa is an open pollinated plant, hence exhibits only a limited amount of variations and offers very little scope of improvement through traditional breeding methods. Mutation induction has become a proven way of creating variation within crop varieties in a short span of time. The construction of ideotype and development of new varieties can be made by modifying the characters of plants which are possible only by the use of morphological mutations. These morphological mutants are useful for the development of improved varieties when they are used in cross breeding programmes (Khursheed and Khan, 2016). In the current scenario, emphasis has been laid on both yield and morphological mutations in M1 generation.

(moisture content: 19.94%; seed size: 0.26 mm ± 1.25 × 0.17 mm ± 0.69) in the year 2014 and maintained in the Net House of Aligarh Muslim University, Aligarh, U.P. (Latitude 27.9135° N, 78.0782° E; soil: sandy loam pH 6.87). Selfed plants were only brought forward in subsequent generations.

The investigation presents a comparative account of the mutagenic effects of EMS alone and in combination with DMSO to improve the yield of N. sativa. MATERIAL AND METHODS

Treatments For the mutagen treatment, ethyl methane sulphonate (EMS) and dimethyl sulfoxide (DMSO) were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India and were used for mutation induction in the present investigation. Dry, dormant and healthy seeds of N. sativa L. were also treated with different concentrations of EMS alone (1%, 2%) and in combination with DMSO (2%) for 6h and 9 h duration. The treatments of chemical mutagens were performed at 18°C±1°C. The pH of the mutagen solution was maintained at 6.8. Initially a leader experiment was conducted to determine the lethal dose, best dose and duration of mutagen. The EMS treated seeds (150 seeds each concentration) were thoroughly washed in running water for 1 h and dried on blotting papers. Thoroughly washed seeds were sown in three replicates for each treatment of the mutagen. Untreated plants were used as control. Physio-morphological studies Germination test was conducted at laboratory conditions in Petri-plates, covered with whatman's filter paper and moistened with sterile distilled water to ensure adequate moisture for seed germination. Emergence of cotyledonary leaves above the soil surface was taken as an index for seed germination under field condition(s). Chlorophyll and carotenoid contents in the extracts of leaves were calculated according to the formula given by Arnon (1949) using spectrophotometer (Spectronic 20D, USA).

Mother seed stock of Nigella sativa L. was procured from National Bureau Plant Genetic Resources, New Delhi, Chlorophyll A (mg g-1 leaf fresh mass) = [12.7(OD663) + 2.69(OD645)] ×

V 1000X W

V 1000X W V Total chlorophyll (mg g-1 leaf fresh mass) = (20.2(OD645) + 8.02(OD663)) x 1000X W Chlorophyll B (mg g-1 leaf fresh mass) = [22.9(OD645) + 4.68(OD663)] ×

Carotenoid (mg g-1 leaf fresh mass)

=

109

7.6 (0D 480 ) - 1.49 (OD d x 1000 x W

109

510

)

´ V

110

Chemical Mutagen affects Pollination and Locule.....

Where, OD645, OD663, OD480, OD510 respectively V W d

JANUARY-JUNE 2017

= Optical densities at 480, 663, 480 and 510 nm = Volume of an extract = Mass of leaf tissues = Length of light path (d= 1.4 cm)

Meiotic analysis Young flower buds were collected during early morning hours and fixed in Carnoy's fluid for conducting meiotic analysis. Flower buds were transferred to 70% alcohol after 24 h of fixation. Anthers were smeared in 1% acetocarmine solution and pollen mother cells were examined under binocular compound microscope (Zeiss, Switzerland) at 40× for their behavior at various stages of microsporogenesis. Pollination and capsule studies Pollination mechanism have observed before and at the time of anthesis of the flowers of the plant during early morning (06:00 a.m. to 11:00 a.m.) and the photographs have been captured by using camera (L340, Nikon Coolpix, Germany). The number of locules per capsule is calculated separately in each treatment at the time of harvesting, when the fruits change to brownish color and also, comparative photographs of the capsules have been captured. Statistical analysis of data collected for various quantitative traits, performing analysis of variance (ANOVA) for significance, Sigma-Plot 10.0 for graphical representation and Duncan‫׳‬s test using SPSS 17.0 software. The leaf area has been estimated by leaf area meter (LA211, Systronic, India). RESULTS AND DISCUSSION Germination, seedling growth and survival rate The data of rate of germination and survival of plants are given in Table 1. Germination rate of control plants was 88.20% and it varied from 84.03% to 88.00% in EMS alone and combination treatments. The percent of seed germination in treated population decreased with the increase in concentration and duration of treatment, when compared with the control. Maximum shift (-4.17) from mean of control have been observed in 9TE2 whereas the minimum shift (-0.20) was calculated in 6TED1 (Table 1). A dose dependent reduction in the biological parameters has been observed earlier (Kumar and Dubey, 1998). Reduction in seed germination (%)

has been attributed to delay or inhibition in physiological and biological processes necessary for germination, which include defective enzyme production (Kumar, 2005) and inhibition of mitotic process (Ananthaswamy et al., 1971). The survival of plants at maturity decreased linearly with an increase in concentration and duration of mutagenic treatments of EMS alone and in combination with DMSO. While the survival of plants at maturity was 86.55% in control, it was 78.58% and 81.51% in EMS and EMS+DMSO, respectively (Table 1). Progressive decrease in the rate of survival of plants with an increase in the doses of physical and chemical mutagens has been reported in Lycopersicon esculentum (Jayabalan and Rao, 1987) and Lathyrus sativus (Kumar and Dubey, 1998). The adverse effects of physical and chemical mutagens on various biological parameters have been reported (Khursheed et al., 2016; Raina et al., 2017) that may be the most probable reason for reduction in the plant servility. Decrease in pollen fertility have been observed in present experiment, the treatment 6TED1 shows convenient efficiency (98.07%) as compared to control (98.53), but the treatment 9TE2 depicted great deviation (93.87) in pollen fertility as shown in Table 1. These results are in agreement with many workers who have also reported a dose-dependent increase in pollen sterility following mutagenic treatments (Kumar and Dubey, 1998). Pollen sterility in control plant was reported negligible and it enhanced in most of the mutagenic treatments. Pollen sterility varied according to the pollen fertility. There was increase in the pollen sterility in both EMS and EMS+DMSO treatments. Meiotic analysis The diversification of yield traits can be achieved through mutagenesis to create new combinations of structural rearrangement, which are rarely obtained by conventional methods. PMC squash preparations revealed 2n=12 chromosomes in control and treated materials (Fig. 1A-G) and several chromosomal abnormalities were found at the different stages of meiotic division. Mutagen treated samples showed abberations in chromosomal configurations with wide

110



111

Table 1: Statistical analysis of comparative effect of mutagens (EMS and EMS+DMSO) on various physiomorphological characters of N. sativa L. Treatments

Seed Germination Mean±SE

Plant Mature Servivability CV(%) Shift Mean±SE

Pollen Fertility

Plant Height

CV(%) Shift Mean±SE

No. of Primary Branches

CV(%) Shift Mean±SE

CV(%) Shift

Mean±SE

CV(%) Shift

CON

88.20±0.13 a

0.25

0.00 86.55±0.23 a

0.46

0.00

98.53±0.18 a

0.32

0.00

50.49±0.17 e

0.57

0.00

5.67±0.22 c

6.70

0.00

6TE1

87.47±0.20 a

0.40

–0.73 84.30±0.26 c

0.55

–2.25 97.03±0.15 b

0.26

–1.50 51.54±0.13 d

0.43

1.05

5.07±0.26 d

8.88

–0.60

6TE2

86.43±0.26 b

0.52

–1.77 83.20±0.17 de

0.36

–3.35 96.17±0.18 c

0.32

–2.36 53.67±0.01 b

0.04

3.18

4.53±0.15 e

5.74

–1.14

9TE1

85.88±0.46 bc

0.97

–2.32 82.54±0.25 e

0.54

–4.01 95.93±0.18 cd 0.32

–2.60 46.51±0.18 h

0.67

–3.98

5.49±0.17 cd 5.28

–0.18 –0.26

9TE2

84.03±0.15 e

0.30

–4.17 78.58±0.14 g

0.32

–7.97 93.87±0.24 f

0.45

–4.66 45.40±0.19 i

0.71

–5.09

5.41±0.17 cd 5.73

6TED1

88.00±0.35 a

1.02

–0.20 85.06±0.29 b

0.34

–1.49 98.07±0.20 a

0.36

–0.46 48.64±0.19 g

0.68

–1.85

8.49±0.16 a

3.30

2.82

6TED2

85.90±0.21 bc

0.42

–2.30 83.61±0.33 cd

0.40

–2.94 96.83±0.18 b

0.32

–1.70 49.58±0.24 f

0.83

–0.91

6.36±0.08 b

2.04

0.69

9TED1

85.03±0.35 cd

0.71

–3.17 82.50±0.21 e

0.26

–4.05 95.53±0.18 de 0.33

–3.00 52.28±0.14 c

0.48

1.79

5.66±0.16 c

4.95

–0.01

9TED2

84.37±0.23 de

0.47

–3.83 81.51±0.23 f

0.28

–5.04 95.00±0.29 e

–3.53 54.53±0.15 a

0.50

4.04

5.14±0.07 cd 2.34

–0.53

0.53

Significant correlation at p£0.05; Values followed by the same letter in the same column are not significantly different at the 95% level according to Duncan's test; SE, standard error; CV, coefficient of variance; Shift, deviation from control mean; CON, control; 6TE1, 1%EMS for 6 h; 6TE2, 2%EMS for 6 h; 9TE1, 1%EMS for 9 h; 9TE2, 2%EMS for 9 h; 6TED1, 1%EMS+2%DMSO for 6 h; 6TED2, 2%EMS+2%DMSO for 6 h; 9TED1, 1%EMS+2%DMSO for 9 h; 9TED2, 2%EMS+2%DMSO for 9 h.

Table 2: Frequency of chromosomal abnormalities induced by EMS alone and EMS+DMSO in N. sativa L. Total No. of Abnormal PMCs Observed

% of Abnormal PMCs

Disturbed Polarity

Multi nucleate

Micro nucleate

Bridges

Laggards

% of Abnormal PMCs

Telophase- I/II

Unequal Separation

Bridges

Laggards

% of Abnormal PMCs

Stickiness

Anaphase-I/II

Stray chromosomes

Metaphase-I/II Precocious Movement

Total No. of PMCs Observed

Multi-valents

Treatments

Total % of Abnormal PMCs Observed

CON

265

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

6TE1

257

-

1

3

1

1.94

-

1

2

1.16

1

-

1

-

3

1.94

13

5.05

6TE2

262

1

2

1

3

2.67

1

2

1

1.53

-

-

1

1

1

1.14

14

5.34

9TE1

271

2

3

2

4

4.05

3

3

2

2.95

1

1

2

1

2

2.58

26

9.60 14.28

9TE2

252

3

5

3

4

5.95

4

2

4

3.97

2

1

2

2

4

4.36

36

6TED1

263

-

1

1

1

1.14

-

1

1

0.76

-

1

2

1

3

2.66

12

6TED2

269

1

1

2

1

1.86

1

2

1

1.49

-

1

1

1

2

1.86

14

4.65 5.20

9TED1

258

-

3

2

3

3.10

1

1

2

1.55

-

1

1

3

1

2.32

18

6.98

9TED2

278

3

5

4

4

5.75

3

4

3

3.60

1

1

2

3

4

4.00

37

13.31

PMCs, Pollen Mother Cells; CON, control; 6TE1, 1%EMS for 6 h; 6TE2, 2%EMS for 6 h; 9TE1, 1%EMS for 9 h; 9TE2, 2%EMS for 9 h; 6TED1, 1%EMS+2%DMSO for 6 h; 6TED2, 2%EMS+2%DMSO for 6 h; 9TED1, 1%EMS+2%DMSO for 9 h; 9TED2, 2%EMS+2%DMSO for 9 h.

spectrum of variations like stickiness, laggards, multivalent, bridges and unequal separation at anaphase I/II and metaphase I/II. The dominant meiotic aberrations at telophase I/II were disturbed polarity, multinucleate and micronuclei condition (Fig. 1 and 2). Similar types of aberrations have also been reported in different plants, such as Plantago ovate (Kumar and Srivastava, 2001), Capsicum annum L. (Anis and Sharma, 1997), Vicia faba (Khursheed et al., 2015). The percentage of abnormal cells at metaphase ranged from 5.95% at 6TE2 to 1.14% at 6TED2 (Table 2). The

frequency of abnormalities at anaphase increased from 0.76% at 6TED1 to 3.97% at 9TE2. The unequal separation and bridges were more frequent than laggards, which were absent in low concentration treatments (Table 2). The result showed that the percentage of abnormal pollen mother cells at metaphase varies from 1.14% to 4.36% at the treatment of 6TE1 to 9TE1, respectively (Table 2). The overall result showed that the treatment of 1%EMS + 2%DMSO for 6 h (i.e. 6TED1) has competent mutagenicity but higher efficiency as compared to other treatments. Also the tetrahedral tetrad

111


112

JANUARY-JUNE 2017

Table 3: Statistical analysis of comparative effect of mutagens (EMS and EMS+DMSO) on various quantitative phenotypic characters of N. sativa L. Treatments

Seed Germination Mean±SE

Plant Mature Servivability CV(%) Shift Mean±SE

Pollen Fertility CV(%) Shift Mean±SE

CON

5.30±0.15 c

5.09

0.00 1.46±0.23 ab

28.08

6TE1

4.63±0.26 de

9.72

–0.67 1.19±0.19 ab

0.00

Plant Height CV(%) Shift Mean±SE

5.00±0.28 bcd 9.60

0.00

74.05±0.89 b

No. of Primary Branches CV(%) Shift

Mean±SE

CV(%) Shift

2.08

0.00

2.23±0.20 ab 15.25

0.00

27.73 –0.27 4.74±0.15 bcd 5.49

–0.26 66.00±0.90 cd 2.36

–8.05

2.03±0.21 b

–0.20

17.73

6TE2

4.34±0.22 de

8.99

–0.96 1.07±0.15 b

23.37 –0.39 4.64±0.22 cd

8.18

–0.36 63.55±0.87 de 2.38 –10.50 1.88±0.14 b

13.30

–0.35

9TE1

4.46±0.12 de

4.71

–0.84 1.05±0.21 b

34.29 –0.41 4.56±0.28 cd

10.75 –0.44 62.75±0.88 de 2.44 –11.30 1.77±0.25 b

24.29

–0.46 –0.51

9TE2

4.23±0.17 e

6.86

–1.07 1.01±0.18 b

30.69 –0.45 4.07±0.38 d

16.22 –0.93 60.17±0.95 de 2.74 –13.88 1.72±0.17 b

17.44

6TED1

8.41±0.16 a

3.21

3.11 1.83±0.30 a

28.42

8.61±0.35 a

9.23

1.61

102.30±0.51 a 0.95

18.25

2.75±0.13 a

8.36

0.52

6TED2

6.22±0.12 b

3.22

0.92 1.45±0.21 ab

24.83 –0.01 5.64±0.38 b

12.33

0.64

75.32±1.73 b

3.97

1.27

2.31±0.38 ab 28.14

0.08

68.13±1.45 cd 3.68

–5.92

0.37

9TED1

5.40±0.23 c

7.22

0.10 1.25±0.20 ab

28.00 –0.21 5.27±0.29 bc

9.49

0.27

1.87±0.18 b

16.04

–0.36

9TED2

4.87±0.16 cd

5.54

–0.43 1.15±0.30 ab

45.22 –0.31 4.54±0.21 cd

8.15

–0.46 63.44±1.02 de 2.27 –10.61 1.74±0.15 b

14.94

–0.49

Significant correlation at p£0.05; Values followed by the same letter in the same column are not significantly different at the 95% level according to Duncan's test; SE, standard error; CV, coefficient of variance; Shift, deviation from control mean; CON, control; 6TE1, 1%EMS for 6 h; 6TE2, 2%EMS for 6 h; 9TE1, 1%EMS for 9 h; 9TE2, 2%EMS for 9 h; 6TED1, 1%EMS+2%DMSO for 6 h; 6TED2, 2%EMS+2%DMSO for 6 h; 9TED1, 1%EMS+2%DMSO for 9 h; 9TED2, 2%EMS+2%DMSO for 9 h.

Fig. 1: Meiotic abnormalities in N. sativa L. Control– (A) Six Bivalents; (B) Metaphase І; (C) Metaphase ІІ; (D) Anaphase І; (E) Anaphase ІІ; (F) Telophase І; (G) Telophase ІІ; Abnormalities– (H) Clumped Metaphase І; (I) Stickiness at Metaphase ІІ; (J) Laggard at Anaphase І; (K) Chromosome Ring; (L) Tri-nucleus condition.

112



113

Fig. 2: (M) Stray Chromosome with Micronuclei; (N) Ring- and Rod-Valents at Anaphase І; (O) Flipping chromosome at Metaphase І; (P) Polyploidy condition; (Q) Giant Nucleolus at Telophase І; (R) Lagging Chromosome Pair at Metaphase ІІ; (S) Disturbed Polarity with Micronuclei; (T) Multiple Bridging; (U) Nucleomics; (V) Normal Tetrahedral Tetrad; (W) T-shape Tetrad; (X) Linear Tetrad.

Fig. 3: comparative representation of pigments- chlorophylls and carotenoids in N. sativa L. with EMS and EMS+DMSO treatments at 6 h and 9 h durations (chl a – chlorophyll a; chl b – chlorophyll b; T. chl – total chlorophyll; crot. - carotenoids).

Fig. 4: comparative representation of leaf area and average leaf number among the treatments of EMS and EMS+DMSO in N. sativa L. (AOL – area of leaf; ANL/P – average number of leaf per plant; TAL/P – total area of leaf per plant).

113

114


JANUARY-JUNE 2017

Fig. 5: 1- Normal Dicotyledon; 2- Monocotyledon; 3- Tricotyledon; 4- Stunted Growth with Early Flowering; 5- Thick Stem; 6- Normal leaflet Notches; 7, 8, 9- Variation in Leaflet Notches; 10- Cup-shaped leaflet; 11- Leaflet Arrangement Changes from Alternate to Opposite; 12- Lax pinnae of Leaflets; 13- Chloroxantha Mutant; 14- Normal Flower; 15- Over-lapped Increase in Petal Number; 16- Cross-pollination Mechanism Changes to Complete Self-pollination; 17- Variation in Flower Color; 18, 19, 20, 21- Locules Number Increases from 5 to 6, 7 and 12.

in control served towards the linear and T-shaped tetrads in various treatments (Fig. 2). Therefore, it provides a considerable clue to assess sensitivity of plants towards different mutagens, and to ascertain the most effective mutagen dose for a given crop. Bio-physiological Damage The estimation of bio-physiological damages caused by mutagens helps in determining the sensitivity of a biological material as well as the potency of a particular mutagen. In the present investigation, the effects of different doses of EMS and DMSO treatments measured in terms of chlorophyll and carotenoid contents at early stages from the treated seeds along with the control were studied. The contents of chlorophyll-a, chlorophyll-b, total chlorophyll and carotenoids were 0.826, 0.537,

1.268 and 0.294 mg/g fresh weight, respectively in control plants. The chlorophyll and carotenoid content decreased in random trend with different doses and duration of EMS alone and in combination with DMSO. The chlorophyll-a, chlorophyll-b and total chlorophyll content increased to 0.890, 0.573 and 1.462 mg/g FW, respectively at 1%EMS+2%DMSO for 9 h, but the carotenoids content deceased to 0.289 mg/g FW at this very particular treatment. There was increase in chlorophyll-a by 7.75% at 1%EMS+2%DMSO for 6h when compared with control. The lowest content of chlorophyll-a (0.736), chlorophyll-b (0.480) and total chlorophyll content (1.216 mg/g FW) was observed at 2%EMS+2%DMSO for 9 h. The increase in chlorophyll content hints towards the increase in the productivity of

114



the crop (Larcher, 1995). The carotenoids content decreased (0.230 mg/g fresh weight) at 1%EMS+2%DMSO for 9h when compared with control (0.294 mg/g fresh weight) (Fig. 3).

in lentil (Amin et al., 2015) and Chlorisguana Kunth (Krishna et al., 1984), with the use of different mutagens.

Effect on leaf type, arrangement and shape: Modifications of leaf arrangement, shape, size and colour are the most useful phenotypic marker in mutation breeding due to their wide appearance and easy detection (Laskar et al., 2015). The chemical mutagens employed in the study resulted in wide range of variations in leaf shape and leaflet arrangement on rachis (Fig. 5). Control plants have pinnately compound and notched leaves with 7-9 leaflets (Fig. 5(6)). Different types of morphological variation have been observed in treated populations, and the prominent was increased in number in notches from 3-5 in leaflets and irregular shapes of notched leaflets (Fig. 5(7-9)). Some leaflets were rudimentary, thick, curved and leathery (Fig. 5(10, 12)). The second dominant morphological variation is the increase in leaf size, number of leaflets and change in phyllotaxy from alternate to opposite in some treatments (Fig. 5(11)). The treatment 1% EMS+2% DMSO for 6 h, resulted the significant increase in the leaf area as well as number of leaflets. The total area of leaf was increased to 370.07 cm2 as compared to control (307.58 cm2), whereas there was decline in the total leaf area by 256.33cm2 at 2%EMS treatment for 9 h (Fig. 4). The significant increase in leaf area (20.31%) at 1%EMS+2%DMSO treatment for 6 h, directly appertain to the increase in photosynthetic activity as well as the productivity of the crop. Earlier results also showed the impact of leaf size and morphology on the productivity of the plant (Laskar et al., 2015). Leaf morphology of Vicia faba (Kumar et al., 1993), Vigna radiate L. (Bhat et al., 2007) with the use of different mutagenic agents, showed similar results. The leaf abnormalities may be due to actual mutation processes which are most easily induced in leguminous plants (Blixt, 1972) or due to chromosomal alterations (Grover and Virk, 1984). A chlorophyll variant chloroxantha have been observed in the treated population (Fig. 5(13)). At the time of germination, s ome plantlets showed only single and some showed three cotyledonary leaves in comparison to the two cotyledonary leaves in control (Fig. 5(1-3)). The other variants have been also observed like rudimentary growth (Fig. 5(4)) and thick stem (Fig. 5(5)), which helps the plant to withstand in drastic conditions. Similar observations have also been reported

115

Flowering physiology The arrangement of petals in flower and also the number of petals varied from 5 to 7 in treated population (Fig. 5(14 & 15)). Normally, the flowers of N. sativa showed cross pollination, however, treated population (especially at 1%EMS+2%DMSO for 6 h), resulted in higher seed yield due to the modified mode of selfpollination. In treated plants the style move downwards, bend at an angle greater than 90◦ towards the anthers, during the time period of 8:00 am to 10:00 am (Fig. 5(16)). As per the reports of Abu-Hammour et al.,(2011) the cross pollination cannot fertilize all the ovules, due to variable number of multi-ovule carpel, less quantity of pollen grains and little volume of nectar which can be attributed to the lower seed set of plant. As per previous reports, N. sativa shows anthesis in morning hours (7am to10am) and synchronization occurs on the last day of male and female stage of flowering (Andersson, 2005). The plant has also a property that the stigma receptivity changes rapidly with slight change in temperature i.e; why artificial selfing falls to seed set. The present change in pollination was accompanied by the increase in locules per capsule that lead to 16.02% increase in plant yield. Thus, the open pollination mechanism has been replaced by complete self-pollination (Fig. 5(16)). By this mechanism the seed set in this crop have increased to the greater extent, without the pollen limitation as well as assure non-essentaility of pollinators. Also, the color of stamens changes towards the blue from pale-yellow (Fig. 5(17)). The variation in flowering physiology has been observed in other plants by the use of physical and chemical mutagens (Hasan et al., 2013; Laskar et al., 2015). Since, flowering like monogenic traits are manipulated through mutation breeding, with independent segregation has efficient potential for ascertaining the invariant expressivity of recessive gene which assist the selection of stable high yielding mutants. The present investigation confirms the mutagenic effects on the pathways of expression of flowering gene. Quantitative trait analysis The unexpected results of quantitative traits like plant height, number of primary branches, number of capsules, size of capsule, number locules per capsule and seed weight of the present experiment, leaning towards the

115

116


improvement in crop yield. As the plant height deviates variably with the different treatments, increases to 54.53cm at 9TED2 in contrast to control (50.49cm), whereas it decreases to 45.53cm at 9TE2 (Table 1). The negative deviation in plant height has been reported in other crops, due to chemical mutagens as well (Gaibriyal et al., 2009; Laskar et al., 2015). The creditable increase in primary branches (8.49) with respect to control (5.64), efforts greatly to increase in the plant fertility (Table 1). The number of capsules increased significantly from 5.30 to 8.41 at the 1%EMS+2%DMSO, 6hrs as shown in (Table 3). The yield attributed traits such as capsule length; locules per capsule; seeds per capsule and seed weight have reflected dose dependent divergence. The treatment 6TED2 have shown positive significant deviation from control population, as the capsule length increased to 1.83cm from 1.46cm, locules per capsule to 8.61 from 5.00, seed per capsule to 102.30gm from 74.05gm and seed weight to 2.75gm from 2.23gm, but the negative deviation of these characters was estimated at 9TE2 (Table 3). The combination treatment showed the significant interesting increase of 6-12 locules per capsule at 1%EMS+2%DMSO for 6h (Fig. 5(18-21)). Similar observation was reported by Prabha et al., (2010) in N. sativa where it showed significant variation in terms of number of branches; seedling height; 1000 seed weight at the 4.5mM SA treatment. It can be inferred that the range of quantitative characters through induced mutations are random, bi-directional and the direction of the mutation depends on the genotype/trait under study and the dose applied. CONCLUSION Based on the present study, it can be concluded that the combined treatment of 1%EMS+2%DMSO for 6h resulted in large variation in the quantitative traits, with minimum biological damage as seen significant increase in the yield. It is suggested that the increase in the yield is due to the modification of pollination mechanism that shifts from cross-pollination to self-pollination and was accompanied with the increase in number of locules per capsule. Other combination treatments showed wide divergent results of physio-morphological and quantitative traits, but lesser cytological aberrations as well. It is, therefore, concluded that the low dose of combination treatment (1%EMS+2%DMSO, 6h) can have a role in the crop improvement in Nigella sativa L. through accretion of seed quantity.

JANUARY-JUNE 2017

REFERENCES Datta A. K., Saha A., Bhattacharya A., Mandall A., Paul R. and Sengupta S. 2012. Black cumin (Nigella sativa L.) – a review. Journal of plant development sciences vol. 4 (1): 1-43. Haddad P. S., Deport M., Settaf A., Chabbi A. and Cherrh Y. 2003. Comparative survey on the medicinal plants more recommended by traditional practitioners in Morocca and Canada. Journal of Herbs Spices and Medicinal Plants. 10: 25-45. Iqbal M. S., Qureshi A. S. and Ghafoor A. 2010. Evaluation of Nigella sativa L., for genetic variation and ex–situ conservation. Pak. J. Bot. 42: 2489-2495. Datta A. K. and Biswas A. K. 1985. Induced mutagenesis in Nigella sativa L. Cytologia. 50: 545-562. Raina A., Laskar R. A., Khursheed S., Amin R., Tantray Y. R., Parveen K. and Khan S. 2016. Role of mutation breeding in crop improvement- Past, Present and Future. Asian Research Journal of Agriculture. 2(2):1-13. Adamu A. K. and Aliyu H. 2007. Morphological effects of sodium azide on tomato (Lycopersicon esculentum Mill). Scientific World J. 2(4):9- 12. Greene E. A., Codomo C. A., Taylor N. E., Henikoff J. G., Till B. J., Reynolds S. H., Enns L. C., Burtner C., Johnson J. E., Odden A. R., Comai L. and Steven H. 2003. Spectrum of chemically induced mutations from a large scale reverse genetic screen in Arabidopsis. Genetics. 164: 731–740. Mendhulkar V. D., Bhati T. and Kharat S. N. 2015. Cytogenetical and Morphological variations in EMS treated Glycine max Linn. (Merr.). Research in Biotechnology. 6(4): 19-26. Poehlman J. M. and Sleeper D. A. 1995. Breeding Field crops. Panima Publishing Corporation. New Delhi, India. 278 p. Khursheed S. and Khan S. 2016. Genetic improvement of two cultivars of Vicia faba L. using gamma irradiation and ethyl methane sulphonate mutagenesis. Legume Research. 14: 1-7. Dixit V., Prabha R. and Chaudhary B. R. 2013. Effects of EMS and SA on meiotic cells and thymoquinone content of Nigella sativa L. cultivars. Caryologia. 66(2): 178-185.

116



Singh S. P., Singh R. P., Singh N. K., Prasad J. P. and Sahi J.P. 2007. Mutagenic efficiency of gamma rays, EMS and its combination on microsperma lentil. Internat. J. Agri. Sci. 3(1): 113-118.

Khursheed S., Laskar R. A., Raina A., Amin R., Khan S. 2015. Comparative analysis of cytological abnormalities induced in Vicia faba L. genotypes using physical and chemical mutagenesis. Chromosome Science. 18: 47-51

Prabha R., Dixit V. and Chaudhary B. R. 2010. Comparative spectrum of sodium azide responsiveness in plants. American-Eurasian J. Agric. & Environ. Sci., 8 (6): 779-783. Arnon D. I. 1949. Copper enzymes in isolated chloroplasts, polyphenol oxidase in Beta vulgaris. Plant Physiol., 2: 1–15. Kumar A. 2005. Role of ethyl methane sulphonate on the germination of Coriandrum sativum L. Int. J. Mendel. 22: 29. Ananthaswamy H. N., Vakil U. K. and Srinivasan A. 1971. Biochemical and physiological changes in gamma irradiated wheat during germination. Rad. Bot. 11: 1-12. Jayabalan N. and Rao G. R. 1987. Effect of physical and chemical mutagens on seed germination and survival of seedling in Lycopersicon esculentum Mill. J. Indian Bot. Soc. 10: 133-137. Kumar S. and 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.)Var. P505. J. Phytol. Res. 11(2): 165-170. Raina A., Laskar R. A., Khursheed S., Khan S., Parveen K., Amin R., Khan S. 2017. Induce physical and chemical mutagenesis for improvement of yield attributing traits and their correlation analysis in chickpea. International Letters of Natural Sciences. 61:14-22 Khursheed S., Raina A., Khan S. 2016. Improvement of yield and mineral content in two cultivars of Vicia faba L. through physical and chemical mutagenesis and their character association analysis. Archives of Current Research International. 4(1): 1-7 Kumar G. and Srivastava U. 2001. Cytomictic variations in Isabgol (Plantago ovate forsk). The Nucleus 44: 180-182. Anis M. and Sharma N. 1997. Induced variation in chiasma frequency in Capsicum annuum L. in response to chemical mutagens.Thai. J. Agric. Sci. 30: 43-52.

117

Larcher W. 1995. Gas exchange in plants. In W. Larcher: Physiological plant ecology. 3rdedition. Pp. 74-128. Berlin: Springer. Laskar R. A., Khan S., Khursheed S., Raina A. and Amin R. 2015. Quantitative analysis of induced phenotypic diversity in Chickpea using physical and chemical mutagenesis. Journal of Agronomy. 14(3): 102111. Kumar S., Vandana and Dubey D. K. 1993. Studies on the effect of gamma rays and DES on germination, growth fertility and yield in faba-bean. FABIS News letter. 32: 15–18. Bhat T. A., Khan A. H. and Parveen S. 2007. Spectrum and frequency of chlorophyll mutations induced by MMS, gamma rays and their combination in two varieties of Vicia faba L. Asian Journal of Plant Science. 6: 558–561. Blixt S. 1972. Mutation genetics in pisum. Agricultural and Horticultural Genetics. 30: 1–293. Grover I. S. and Virk G. S. 1986. A comparative study of gamma rays and some chemical mutagens on the induction of chromosomal aberrations in mung bean (Vigna radiata L. Wilczek). Acta Botanica Indica. 14: 170–180. Amin R., Laskar R. A., Khan S. 2015. Assessment of genetic response and character association for yield and yield components in Lentil (Lens culinaris L.) population developed through chemical mutagenesis. Cogent Food and Agriculture. 1: 1000715. Krishna G., Shivashankar G., & Nath J. 1984. Mutagenic response of Rhodes grass (Choris gayana Kunth.) to gamma rays. Environmental & Experimental Botany. 24: 197–205. Abu-Hammour K.A. and Wittmann D. 2011. Pollination of Nigella sativa L. (Ranunculaceae) in Jordan Valley to improve seed set. Advances in Horticultural Science, Vol. 25(4): 212-222. Andersson S. 2005. Floral costs in nigella sativa

117

118


JANUARY-JUNE 2017

(ranunculaceae): compensatory responses to perianth removal. American Journal of Botany. 92(2): 279–283.

of quantitative traits with grain yield in bread wheat (Triticum aestivum L.). Asian J. agric. sci. 1(1): 4-6.

Hasan M. T. and Deb A. C. 2013. Inheritance of double flower per peduncle and flower colour in chickpea (Cicer arietinum L.). Electron. J. Plant Breed. 4: 1228-1231.

Siddiqui M. A., Khan I. A. and Khatri A. 2009. Induced quantitative variability by gamma rays and ethylmethane sulphonate alone and in combination in rapeseed (Brassica napus L.). Pak. J. Bot. 41(3):118995.

Gaibriyal M., Kumar L. B., Upadhyay A. and Upadhyay R. 2009. Genetic variability and association

118