1837 EFFECT OF SODIUM AZIDE ON THE GROWTH OF CAPSICUM ...

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Pak. J. Biotechnol. Vol. 9 (1) 13- 20 (2012)

ISSN 1812 -1837

EFFECT OF SODIUM AZIDE ON THE GROWTH OF CAPSICUM ANNUUM (CHILI) M. Umar Dahot, M. Rafiq, Ambreen M. Arif and S. H. Ahmed Naqvi Plant Biotechnology Research Laboratory, Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro, Sindh, Pakistan ABSTRACT The present study was carried out to check the mutagenic effects of 0.5, 1.0 and 2.0% NaN3 on the germination and growth of Capsicum annum (Chili). The results showed that 0.5% NaN3 was most appropriate for the creation of mutagenesis and was not highly lethal. The higher germination rate was observed in control and 0.5% NaN3 treated seeds but germination rate was decreased in 1.0% and 2.0% NaN3 treatment. The variation was observed in plant fresh and dry weight, total and reducing sugars and total protein contents of plants in different concentrations of NaN3 treatments.

INTRODUCTION Green chilies are one of the most important commodities used as a vegetable, spice, medical herb, and ornamental plant by billions of people every day. The green chilies are full of flavour with bright green colour. The chilies plant belongs to the genus Capsicum order Solanales and family Solanaceae Kingdom planatae and species annuum, and scientific name is Capsicum annuum. The common names are green chilies, Capsicum, Chili pepper, hot pepper, cayenne, red pepper, sweet pepper, green pepper, bell pepper and Tabasco pepper. Capsicum contains approximately 20 to 27 species. Capsicum annuum, Capsicum baccatum, Capsicum chinense, Capsicum frutescens and Capsicum pubescens are cultivated in different parts of the world. Among the five species of cultivated, Capsicum annuum is one of the most common cultivated crops worldwide (Tong and Bosland, 1999) followed by Capsicum furtescens (Bosland and Votava, 2003). Green chilies are comprises numerous

chemicals including steam volatile oils, capsaicinoids, caroteniods, vitamins, protein, fiber and mineral elements (Votava and Tomas 2003, Andres, 2003). Chilies are low in sodium and cholesterol free, rich in vitamins A, C and good source of potassium folic acid and vitamin E. Fresh green chili pepper contain more vitamin C than citrus fruits and fresh red chili. Chili has more vitamin A than carrots (Osuna and Wall, 1998). Two chemical groups produced by chili are capsaicinoids and caroteniods. The capsaicinoids are alkaloids that make hot chili pungent. A large number of carotenoids provide high nutritional value and the color to chili (Britten and Hornero, 1997). The chili pepper, a hotly pungent variety of Capsicum was first cultivated by the people of Central and South America in around 3000BC. Columbus brought seeds back to Europe in 1493, and from there it has spread to the cuisines of the entire world. In Pakistan, only two species like Capsicum annuum and Capsicum frutescens are known and most

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of the cultivated varieties belong to the species Capsicum annum. In Pakistan, it is a significant cash crop occupying 19% of the total area under vegetable cultivation mainly concentrated in Sindh (46917 ha), Punjab (6400 ha), Baluchistan (2082 ha) and NWFP (392 ha). The climatic conditions of Pakistan are diverse, chilies are grown at different times in different ecological zones in each province (Shah et al., 2008; & 2009). Pakistan is the fifth largest chili producing country in the world, according to the Planning and Development Department, but when it comes to export it stands at number 21 due to progressive decrease in area and production of chili in Pakistan. Since 1999-00 to 2004-05, the area under chili crop has decreased from 86.8 to 48.7 thousand hectares and production from 115.5 to 90.5 thousand tones. The aim of present work was to check the effect of sodium azide on morphological and physiological characters as well as on biochemical of Capsicum annum MATERIALS AND METHODS The present research work was carried out in Plant Biotechnology Research Laboratory, Institute of Biotechnology and Genetic Engineering (IBGE), University of Sindh Jamshoro. All chemicals were used of analytical grade purchased from Fluka, Merck and BDH. The reagents were prepared in distilled water. Selection and sterilization of green chilies seeds for mutagenesis: Mature seeds of green chilies were obtained from the local market of Hyderabad. Its sterilization was performed under aseptic condition in laminar air flow cabinet. The seeds were sterilized in 2% sodium hypo chloride for 30 minutes with continuous

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shaking. Afterward the seeds were rinsed three times with sterile distilled water and kept under aseptic conditions. Treatment of seeds with sodium azide: Seeds were treated with sodium azide solution at concentrations 0.5%, 1% and 2% for 1 ½ hr. Sodium azide solutions were prepared in 20ml distilled H2O. In each treatment, seeds were soaked in 20ml of the mutagenic solution. The control seeds were soaked in distilled H2O, washed periodically and agitated at room temperature. The seeds were washed with tap-water to remove excess mutagens and dried. The treated and control seeds (50 seeds per treatment) were sown in the pots according to RCBD and experiment was repeated two times. Analysis of morphological characters: The plants were harvested after 2nd, 4th and 6th week of germination and data of germination was collected. The germinating seedling height, roots length, shoot length, fresh and dry weight was recorded. Plant Height: Height of plant was measured in centimeter from root tip to the shoot tip point, with the help of measuring (inch) tape/meter. Shoot length & root length were also measured. Fresh weight: The harvested plant parts were washed with tap water. After drying the plant on filter paper, the roots, shoots and leaves were separated and determined the fresh weight Dry weight: The separated leaves, roots, and shoots were dried in oven at 60°C for 48hrs and then dry weight was determined. Biochemical Analysis: Chlorophyll concentration was analyzed by the method of Shabala et al., (1998) whereas total sugar content was measured according to the method reported by Montgomery (1961). Reducing sugar contents from test

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sample were determined by the method of Miller (1959) and the protein concentration was checked according to the Lowry et al., (1951) method. RESULTS In the present study, the effect of different concentration of sodium azide (0.5%, 1%, and 2%) was checked on green chilies seeds growth. Fifty seeds of each concentration as well as control were sown in pots. The plants were germinated after two weeks. It was observed that 75% seeds were germinated in the control (seeds not treated with NaN3) and 0.5% NaN3 treated seeds. Similarly 62.5% seeds were germinated, which were

treated with 1% and 2% sodium azide. It means that the germination rate decreased in 1% and 2% NaN3 treatment as compared to control and 0.5% NaN3 treatment (Table-1). It was observed that plant height was decreased in 2% NaN3 treated plants. However, the mean value of plant height was found 8.0 cm in 1% NaN3 treated plants and control plants. It was noted that the lowest mean value (7.0 cm) of plant height was observed in 2% NaN3 treated plants. It means that 2% NaN3 treatment has the reducing effect on growth while 0.5% NaN3 treatment is suitable for increasing of plant height (Table-1).

Table-1: Germination rate and height of Chili seeds Plant height Plant height Plant height % (cm) (cm) (cm) Treatment germination nd th th 2 Week 4 Week 6 Week Control

75

5.666 ± 2.33

6.166± 2.31

0.5% NaN3 75 5.5 ± 2.07 7.0± 2.44 1% NaN3 62.5 4.8± 2.16 6.6± 2.07 2% NaN3 62.5 3.4 ± 2.07 5.2± 1.92 Mean value of randomly selected 12 plants The total mean value of fresh weight of control plant leaves was found to be 1.757 g. The highest fresh weight of leaves (1.869 g) was observed in 0.5 % NaN3.The lowest leaves fresh weight (1.7404 g) in 2% NaN3 treated plants. The highest stem fresh weight (2.036g) was observed in 1% NaN3 treated seeds. The lowest stem fresh weight (1.7825 g) was

8.0± 1.89 8.5± 3.03 8.0± 1.87 7.0± 1.41

noted in 0.5% NaN3 treatment. In control fresh weight of stems were observed 1.840g as shown in Table-2. The total mean fresh weight of roots in control plants was noted 1.648 grams. The highest fresh root weight was observed in 2% NaN3 treatment. Whereas lowest roots fresh weight (1.591 g) was observed in 1% NaN3 treatment (Table-2).

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Table-2: Results of fresh weights of different parts of Chilli plant Treatment control 0.5% 1.0% 2.0%

Leaves (grams) 1.757 ± 0.045

Stems (grams) 1.840 ± 0.027

Roots (grams) 0.191 ± 0.027

1.869 ± 0.042

1.782 ± 0.053

0.13 ± 0.0070

1.813 ± 0.119

2.036 ± 0.441

0.183 ± 0.0175

1.7404±0.0182 1.840 ± 0.012

0.134 ± 0.010

Mean value of randomly selected 12 plants The total mean values of dry weight of control plant leaves were found to be 0.191g. The lowest leaves of dry weight (0.13 g) were noted in 0.5% NaN3 treated plant. The highest stem dry weight (0.152 g) was observed in control without treated seeds. Whereas, lowest stem dry weight (0.099 g) was noted in 0.5% NaN3. In control dry weight of

stems was observed 0.229g. The total mean dry weight of roots in control plants was noted 0.229 grams. The highest dry root weight was observed (0.229 g) in control plant seeds. Whereas lowest roots dry weight (0.1249 g) was observed in 0.5% NaN3 treatment as shown in Table- 3.

Table-3: Results of dry weights of different parts of Chilli plant Treatment Control 0.5% 1.0% 2.0%

Leaves (grams) 0.191 ± 0.027

Stem (grams) 0.152 ± 0.043

Roots (grams) 0.229 ± 0.071

0.13 ± 0.007

0.099 ± 0.021

0.124 ± 0.029

0.183 ± 0.017

0.141 ± 0.028

0.215 ± 0.053

0.134 ± 0.010

0.126 ± 0.010

0.143 ± 0.0115

Mean value of randomly selected 12 plants The Chlorophyll contents present in leaves of control plant was higher than sodium azide treated plants. Control leaves showed the total chlorophyll 3.205, which was higher as compare to sodium

azide treated plants and the lowest chlorophyll contents 2.399 µg/g fresh weight were calculated in leaves of 0.5% treated plant. The highest contents of chlorophyll a 1.881µg/g, b 1.324µg/g and

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total chlorophyll contents 3.205µg/g were found in control leaves. These results show that sodium azide treatment

decreases the total chlorophyll contents of the leaves in all treatments (Table-4).

Table-4: Results of chlorophyll contents from fresh leaves of plants. Treatment Chlorophyll a Chlorophyll b Mean± SD Total (µg/g) (µg/g) Chlorophyll a+b (µg/g) Control 1.881 1.324 1.602 ± 0.393 3.205 0.5% NaN3 1% NaN3 2% NaN3

1.575 1.669 1.575

0.824 0.826 0.931

1.199 ± 0.531 1.247 ± 0.596 1.253 ± 0.455

2.399 2.495 2.52

Mean value of randomly selected 12 plants The highest value 15.65mg/ml of total sugar contents were found in control stem plants and the second highest value 13.52mg/ml of total sugar observed in 2% sodium azide treated plants of root and lowest value 0.199mg/ml observed in 0.5% sodium azide treated plant roots as

shown in figure -5. The results show that the treatment of 0.5% sodium azide can decrease the total sugar contents in green chilies plant, whereas total sugar content was also decreased in plant leaves and stems treated with 2% sodium azide.

Table- 5: Total sugar contents in different parts of Chilli plants Treatment

Leaves (mg/ml)

Stem (mg/ml)

Roots (mg/ml)

Control

1.338 ±0.0494

15.65 ± 0.197

0.705 ± 0.0452

0.5%

0.656±0.0049

0.519±0.0282

0.199 ± 0.0169

1.0%

1.653 ± 0.1074

0.994 ± 0.0664

0.682 ± 0.124

2.0%

0.605 ± 0.0254

0.432±0.0622

13.52 ± 0.7495

Mean value of randomly selected 12 plants It was observed that highest mean value of reducing sugars was observed in 0.5% sodium azide treated plant that was 0.38mg/ml and the second highest value of reducing sugar was observed in control

stem plant that was 0.32mg/ml and the lowest value of reducing sugar was observed in 0.5% sodium azide root treated plants that was 0.035mg/ml as shown in figure -6

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Table- 6: Reducing sugar contents in different parts of Chilli plants Treatment

Leaves (mg/ml) 0.24 ± 0.0565 0.062 ± 0.0042

Stem (mg/ml) 0.32 ± 0.0565 0.38 ± 0.0141

Control 0.5% 0.06 ± 0.0056 1.0% 0.042 ± 0.0042 0.092 ± 0.0042 2.0% 0.15 ± 0.0042 Mean value of randomly selected 12 plants The highest value of total protein 0.95mg/ml was observed in 2% sodium azide treated plant of leaves the second highest value 0.54mg/ml was observed in control plant roots and the lowest value of

Roots (mg/ml) 0.3 ± 0.0282 0.035 ± 0.0028 0.15 ± 0.0084 0.13 ± 0.0056

total protein 0.08mg/ml was observed in 0.5% sodium azide treated plant roots. The result shows that the 2% sodium azide treatment enhances the protein content’s as shown in Table -7.

Table- 7: Total soluble protein contents in different parts of Chilli plants

Control

Leaves (mg/ml) 0.032 ± 0.0056

0.5%

0.32 ± 0.0424

Stem (mg/ml) 0.19 ± 0.0014 0.36± 0.0424

1.0%

0.08 ± 0.0056

0.058± 0.0028

2.0%

0.95 ± 0.0848

0.17± 0.00141

Treatment

Roots (mg/ml) 0.54± 0.0707 0.08 ± 0.00707 0.13± 0.0049 0.09± 0.0021

Mean value of randomly selected 12 plants DISCUSSION The application of chemical mutagens in improving the quality, yield and economy of the plant is well characterized. The seeds have high regenerative potential and are advantageous for use in mutagenesis. Sodium azide is highly soluble in water, but fewer number of hydrozoic ions are produced in water at low pH. The pH value of soaking solution affects the efficiency of mutation. In present experiment we used different sodium azide concentrations 0.5%, 1% and 2% to observe their effect on germination rate,

shoot length, root length, fresh weight and dry weight, chlorophyll contents, total and reducing sugar contents, total protein contents and compare these results with control plants which were not treated by sodium azide. We observed that the highest seed germination rate (75%) was achieved in control seeds and 0.5% NaN3. It was also found that the highest shoot length was observed as same in 0.5% NaN3 and control plant in second week. However, 1% NaN3 treatment slow down the growth rate of shoots length but after fourth weeks, in 1% NaN3 treated plant

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the shoot length was higher than the control plants. It was noted that the high root length was observed in control plants while the lowest root length was observed in 2% NaN3 treated plant. These results are in the similar range with the results of Asli et al., (2006) in case of sodium azide effect on in-vitro development of four Pea (Pisum sativum L.) cultivars. The chlorophyll contents of the plant leaves checked and highest chlorophyll a (1.881µg/g) and b (1.324µg/g) was observed in control plants and sodium azide treatment was not beneficial for chlorophyll contents. The highest mean value of sugar content were present in control plants and calculated as 15.65mg/ml and the higher mean value of total protein was calculated as 0.95mg/ml in 2% NaN3 treated plant and finally the highest mean value of reducing sugar was calculated as 0.92mg/ml in 2% NaN3 treated plant. Fahad Al-Qurainy in 2009 studied the effects of sodium azide on growth and yield traits of Eruca sativa L. He developed the seedlings from treated seeds with sodium azide at concentration ranged from 1x10 to 5x10 M and these concentrations showed wide -variation in plant growth, plant height, leaf area, fresh and dry weight. It was found that the total chlorophyll content was profoundly affected at all studied concentrations and reduction was found more as compared to control plants and the reduction in chlorophyll content was dose dependent in plants of treated seeds. Ilbas et al. (2005) used NaN3 (SA) to check the morphological and cytogenetic effects of on barley seedling. It was observed that the germination rate affected by increasing the concentrations of NaN3. The length of the roots and leaves were

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affected by treatment with NaN3 on day 14 of the germination period and the mitotic index decreased compared to the untreated control. It is reported that generally sodium azide is very effective in inducing mutations with respect to germination percentage, root length, seedling height and seedling survival in tomato. Sodium azide therefore could be utilized to increase variability in tomato that ultimately increases the possibility of isolating beneficial mutants for improvement of tomato production (Adamu et al., 2007). CONCLUSION The application of sodium azide on crop is easy and inexpensive. It creates mutation to improve their traits. The mutagenic effect of sodium azide appears after sowing the seeds observed by naked eyes. Sodium azide has been used in various crops to improve their yield and quality traits and create resistance to them against harmful pathogen. Different concentration of sodium azide 0.5%, 1% and 2% are used with control in green chilies seeds. The best growth is obtained in 0.5% NaN3 concentration and beneficial growth is obtained in control and reduced growth was obtained in 1% NaN3 concentration. It was observed that these concentrations increases or decreases the growth rate of plant. Total sugar, reducing sugar and total of protein test were done, amongst them the best result of total sugar was observed in control plants root that is 15.65mg/ml. Total protein was higher in 2% NaN3 concentration. Sodium azide should be used to create mutation in those crops, which are highly susceptible for harmful pathogens and made them economically inexpensive and beneficial for farmers.

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