Mutagenic effect of sodium azide ( NaN 3 ) on M 2 generation of Brassica napus L . ( variety Dunkled )

The present study was aimed to investigate the effect of different doses (0.2%, 0.4%, 0.6% and 0.8%) of sodium azide (SA) on M2 generation of Brassica napus (variety Dunkled) on some qualitative, quantitative and biochemical parameters. The result showed that days to germination, days to flowering and days to siliqua maturation were delayed (9.4, 98 and 153 days respectively) in higher treatment as compared to control (5.4, 96 and 147 days respectively). Higher concentration of SA (0.8%) decreased germination percentage (88.56%), plant height (97.95 cm), stem diameter (3.49 cm), number of branches/plant (3.49), number of leaves/plant (10), number of siliqua/plant (64.68), number of seed/siliqua (20.09) and 1000 seeds weight (3.91g) as compared to control. An increase was noticed for siliqua length (5.42 cm) in higher concentration of SA (0.8%) as compared to control (5.24 cm). Proximate analysis showed a significant decrease in oil, oleic acid and glucosinolates contents percentage (42.6%, 54.9%, and 58.5 μmol/g respectively) in higher concentration of SA (0.8%) as compared to control (44.7%, 58.1% and 71.3 μmol/g respectively) while a significant increase was noticed in proteins, moisture, linoleic acid and erucic acid percentage (28.4%, 5.9%, 9% and 37.3% respectively) in higher concentration of SA (0.8%) as compared to control (26.8%, 5.5%, 8.4% and 35.2% respectively.


Materials and methods
The seeds of M2 generation of Brassica napus variety Dunkled were obtained from the Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture, Peshawar.They were presoaked in distilled water for 4 to 6 hours at temperature 25 0 C. Sodium azide solutions of four different concentrations were prepared i.e. 0.2%, 0.4%, 0.6%, and 0.8%.Seeds were soaked in all these different concentration of solution for 5 hours.The control seeds were soaked in distilled water for 5 hours.Sowing Seeds of M2 generation of Brassica napus (variety Dunkled) were sown in the green house of botanical garden, Islamia College, Peshawar on 24 th November 2014.The treated seeds of each dose were sown in 5 pots while seeds of control were sown in separate 5 pots.The seedling survival, growth, morphological changes and seeds setting were carefully observed.

Morphological parameter
Various agronomic and morphological parameters were measured during the plants life span.It includes the following: Days to germination, germination percentage, days to flowering, plant height, number of branches /plant, number of siliqua/plant, number of seeds/siliqua, number of leaves/plant, siliqua length, days to siliqua maturation and 1000 seeds weight Biochemical parameter Oil contents, protein contents, moisture contents, glucosinolates, oleic acid, linoleic acid and erucic acid Statistical analysis Experiment was set up in randomize complete block design with five replica per treatment.Data were statistically analyzed to find out coefficient of variation for each parameter.Two tests were performed i.e. analysis of variation (ANOVA) and least significant difference (LSD) at α = 0.05 using Statistics 10.0 software.

Proximate analysis
Proximate analysis of seeds was carried out at national institute of food and agriculture (NIFA), Peshawar, Pakistan.

Results and discussion
Mutation is a change in a genetic sequence at small level like substitution of a single DNA building block or nucleotide base with another nucleotide base.Meanwhile larger mutations can affect many genes on a chromosome along with substitutions; mutation can also be caused by insertions, deletion or duplications of DNA sequences.In the current study, results of the parameters studied are given below.

Germination
The result of table 1 and figure 1 show the effect of various concentration of sodium azide on days to germination in M2 generation of Brassica napus L. The mean value showed that higher concentration of sodium azide may delay the germination.Days to germination increased slightly at 0.2%, 0.4%.0.6% and 0.8% are (6.4,7.8, 8 and 9.4 days respectively) as compared to control (5.4).The reduction in seed germination in mutagenic treatment had been explained due to delayed or inhibition of physiological and biological processes for seed germination including enzyme activity and inhibition of mitotic process [17, 18].These results are also in the agreement with the finding of [19] on Lycopersicun esculentum and [20] on Arachis hypogaea.

Germination percentage
The results showed that there is no significant effect of various concentration of sodium azide on germination percentage.The lowest mean value for germination percentage were recorded at 0.6%, 0.8% (91.42% and 88.56 respectively) while the highest mean value were recorded at 0.2% and 0.4% (97.142% each) as compared to control (94.28%).This study also confirms the finding of [18], [21-27] who reported that germination percentage, shoot height, root depth, leaflet areas, chl a, chl b and the initial level of fluorescence (F0) decreased with NaN3 treatment in pea.The present result is also in agreement with the finding of [28] who observed almost adverse effect of higher concentration of sodium azide on germination percentage (Table 1 and Figure 1).

Plant height
The data in table 1 and figure 1 show that mean value of plant height was increased (107.5 cm and 110.   2 show average number of siliqua/plant at various concentration of sodium azide on M2 generation of Brassica napus L. In control number of siliqua/plant was 78.78, and increase was noticed at 0.2% (82.77 siliqua/plant).Then decrease in the mean values for number of siliqua/plant in 0.6% (71.36%) and 0.8% (64.68 siliqua/plant) as compared to control.A decrease of siliqua was noticed in 0.4% (66.40 siliqua/plant) as compared to control and other doses.In 0.4% treatment a bifurcated siliqua was also obtained.[11] showed that siliqua/plant was enhanced by gamma rays and EMS (Ethyl methane sulphonate).This was also confirmed by However, present study disagrees with the finding of [41] who noticed increase in number of pods/plant with higher concentration of sodium azide.

Number of seed/siliqua
The data of table 2 and figure 2

Chemical analysis of seeds
The qualitative analysis of seeds was carried out using NIRS (Near Infra-Red Reflectance Spectroscopy) which works on the principle of measuring the absorption of light energy or wavelengths of each component at its characteristic frequency in the near IR region.

Oil percentage
Table 3 and figure 3 show the effect of various concentration of sodium azide on oil percentage in M2 generation of Brassica napus L. The data obtained revealed that the oil content decreased significantly with high concentration of sodium azide.The lowest oil contents were recorded at 0.6%SA and 0.8% SA (43.9% and 42.6% respectively) as compared to control (44.7%) and other treatments.Our results also confirmed the findings of [11].
Introduction Rapeseed (Brassica napus) is also known as rape and Rapa.It is a bright yellow flowering annual herb of family Brassicaceae/ Cruciferae.The family comprised of over 338 genera and about 3,710 species distributed worldwide [1].The family is cosmopolitan in nature but it is one of the cultivated medicinal food plants in middle Asia, North Africa and West Europe [2].Rapeseed cultivars show significantly different responses depending on weather conditions to different irrigation levels [3].The increase of temperature has a negative effect on seed yield of spring oil rape seed, nitrogen show significant effect on seed yield and flowering, phosphorous concentration is not important while potassium concentration effect on the contrary [4].Rapeseed and mustard is the important crop of Brassica group grown as oilseed crop in Pakistan.Oilseeds are rich in proteins (36-40 %) and in addition they contain a high level of fat contents (40-45%) [5, 6].Rapeseed (Brassica napus L.) is now the third most important source of edible oil in the world after soybean and palm oil [7].In Pakistan, after cotton it is the second most important source of oil.It contributes about 17% to the domestic production of edible oil [8].In Pakistan during the year 2010-11 total cultivated area under rapeseed was 439 thousand acres producing 157 thousand tons of seed, which yielded 50 thousand tons of oil while in 2011-12¸ the total cultivated area under rapeseed cultivation was 575 thousand acres, producing 203 thousand tons of seed, which yielded 61 thousand tons of oil [9].Brassica vegetables have been related to the prevention of cancer and degenerative diseases, owing to their glucosinolates and phenolic contents [10].Combination of physical and chemical mutagen show enhancing effect on different parameter of Brassica napus [11].One hundred and sixty three cultivars of annual oil seed crops developed using induced mutations.The maximum number of cultivars have been released in soybean (58), followed by groundnut (44), sesame (16), linseed (15), rapeseed (14), Indian mustard (8), Caster bean (4), white mustard (3) and sunflower (1) [12].During the past seventy years, worldwide more than 2250 varieties have been released that have been derived either as direct mutants or from their progeny [13] and according to FAO/IAEA mutant varieties database there are 2541 mutant cultivars, 30 of which are of Brassica.Among the different breeding method induced mutation has been extensively and successfully used for genetic improvement of any yield attributes either qualitative or quantitative in nature [14].Chemical mutagenesis is considered as an effective mean in improving the yield and quality trait of crop plants [15].Sodium azide (NaN3) is one of the most powerful chemical mutagens used to induce mutation in crop plants [16].The present study was carried out to investigate the effect of sodium azide induced mutation on the qualitative and quantitative characters of Brassica napus L.

Figure 2 .
Figure 2. Illustrates the effect of various concentration of sodium azide on siliqua length [32].Higher concentration of sodium azide reduced siliqua/plant.Similar finding were obtained by [26, 40].
represent the effect of various concentration of sodium azide on number of seed/siliqua in M2 generation of Brassica napus L. Number of seed/siliqua was significantly affected by chemical treatment.The highest mean value for number of seed/siliqua was recorded at 0.6% (22.74 seed/siliqua) and the lowest mean value for number of seed/siliqua was recorded at 0.8% (20.09) as compared to control (22.63).Number of seed/siliqua at 0.2%, 0.4% is 20.43 seed/siliqua and 21.60 seed /siliqua respectively.The result revealed that the number of seed/siliqua was not improved by chemical treatment in M2 generation of Brassica napus L. Similar results was obtained by [42] on Sesame indicum and [27] on Arachis hypogaea L. Increased number of seeds at 0.7% was also recorded by [32].1000 seeds weight Table2and figure2show the effect of various concentration of sodium azide on 1000 seed weight in M2 generation of Brassica napus L. The mean value showed that there is no significant effect of sodium azide on 1000 seeds weight in gram.The weight was maximum at control (4.13g) and minimum at 0.8% (3.91g) as compared to other treatment i.e. 0.2% (3.96g), 0.4% (4.04g) and 0.6% (4.01g).Result showed that various concentration of sodium azide adversely effected 1000 seed weight.Present study is dispute with the finding of [20] who observed an increase in 1000 grain weight at different concentrations.[32] also recorded an increase in 1000 grain weight at higher treatment.Present study also contradict finding of [43, 44].

Table 1 . Effect of various concentration of sodium azide on some morphological characters in M2 generation of Brassica napus L.
A. Days to Germination, B. Germination %age, C. Plant height (cm), D. Stem diameter (cm), E. Number of branches, F. Number of leaves, G. Days to flower initiation, H. Days to flower completion

Table 2 . Effect of various concentration of sodium azide on yield production in M2 generation of Brassica napus L.
height (97.95 cm).The work of [17] also showed almost the same results on stem height which was reduced by higher concentration of sodium azide.Similar results were also obtained by [22], [29-33].