A us tralian Journal of Bas ic and A pplied Sciences , 3(4): 3081-3087, 2009

ISSN 1991-8178

Mutagenic Effect of Sodium Azide on Seed Germination of Eruca sativa (L.)

Salim Khan, Fahad Al-Qurainy

Department of Botany and Microbiology, College of Science, King Saud University, Riyadh,
Kingdom of Saudi Arabia, 11451.

Abs tract: Eruca sa t i v a is a very important crop of M adeterranean region, and highly us ed as s alad
in European countries . Sodium azide (NaN3 ) is a chemic a l mutagen, and widely us ed in crops to
improve their yield and quality traits . W e s tudied the effect of various concentrations of NaN3 ranged
(1mM , 2mM , 3 mM , 4 mM and 5 mM ) on germination and s eedling growth of Eruca at various time
intervals . The s eeds treated at 5 mM of NaN3 , the percent germination profoundly affe c t e d o n d a ys
9 and 12 following its application for 120 min and 180 min of time intervals . The h ig h e s t and lowes t
% germin a t io n was found for 30 min and 180 min of time intervals , whils t s eeds were treated at s ame
concentration of NaN3 . The radicle and coleoptile length were decreas ed as the concentration of NaN3
increas ed, and highly affected at concentratio n s 3 mM , 4 mM and 5 mM res pectively. M ore variation
was found on radicle length than that of coleoptile leng t h a t s a me c o n centrations and at s ame time
intervals .

Key words : Eruca sativa, germination, mutagen, s odium azide,

INTRODUCTION

Eruca sativa is a native of s outhern Europe and central A s ia, where it has been cultivated s ince c la s s ical
times . It is not a commercial crop in the UK or northern Europe, but is widely grown in kitchen and market
gardens in s outhern France, Italy, Greece and the near Eas t, where it is us ed for flavoring s alads . The plant
is naturalized in was te p la c e s , road s houlders and fallow fields in northern and W es tern Europe, well beyond
its original range. It is als o s ometimes referred to as rocket, true rocket, rocket s alad, arugula, roquette or white
pepper. The young plants are us ed as a s alad, vegetable and as gre e n fo d d e r. T ender leaves are reported to
have s timulant, s tomachic, diuretic and antis corbutic activity (Bhandari and Chandel, 1966). The s tudy on
chemical mutagencity on E. sativa is limited in literature, and becaus e thes e mu t a g e n s p la y important role to
improve agronomic traits of plant and als o produce res is tance to them agains t biotic an d abiotic s tres s es . The
s eeds are goo d e xp la n t s fo r chemical mutagens to create mutations in a genome of a cell. A fter treatment of
chemical mutagens , s eeds s how the effects of mutagen as modified morphologica l traits from dis turbed
phys iological proces s es . Germination is the proces s by which a s eed initiates g rowth after a period of
quies cence. It requires s eed imbibitio n , a n d in a s trict s ens e, is defined as the proces s leading to emergence
of th e ra dicle through the tes ta, a tis s ue of maternal origin that s urrounds the embryo (Bewley, 1967;
Koornneef et al. 2002). Germination is thus finis hed o nce the radicle has emerged. Imbibition, i.e. water uptake
by the s eeds , is accompanied by cell expans ion, cell wall s ynthes is , and activation of metabolis m.
A ccumulating evidence indicates that, in general, cell divis ion occurs following germination (de Cas tro et al.
2000; Barroco et al. 2005). The increas e in cell growth that is required for germination is due to cell
elongation. In a very s hort time in t e rv a l, a limited number of cells elongate and go through differentiation
proces s es bas ed on rapid metabolic changes preceding ce ll d iv is ion. The proces s of germination is under the
control of environmental and hormonal factors thus making the s ys tem appropriate for the s tudy of plant
development and the cellular res pons es to thes e factors . In laboratory, the germination depends upon a number
of factors s u c h a s temperature, pH of the s olvent, duration of s oaking etc. Chemical mutagens are the one
caus e of mutation in livin g organis m. Thes e mutagens affect the germination proces s in s eeds . The percent
germination in s eeds depends on the nature of the mu t a g e n and its treatment dos e. M any of thes e mutagens
have clas togenic (chromos ome damaging) e ffe c ts on plants via reactive oxygen-derived radicals (Yuan, 1993).
Chemical mutagen generally produce induced mutations , which lead to bas e pair s ubs titution es pecially GC6A T

Corresponding Author: Salim Khan, Department of Botany and M icrobiology, College of Science, King Saud University,
Riyadh, Kingdom of Saudi Arabia.
Ph-+9664675876 Fax-+9664678533
Email: salim0041@rediffmail.com
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 Aust. J. Basic & Appl. Sci., 3(4): 3081-3087, 2009

(guanine : cytos ine 6 adenine : t h y mine) res ulting in amino acid changes , which change the function of
proteins , but do not abolis h their fu n c t ions as deletions or frame s hift mutations mos tly do (Van der Veen,
1966). Thes e chemomutagens also induced a broad variatio n o f mo rphological and yield s tructure changes in
comparis on to normal plants .
Sodium azide (NaN3 ) is a common laboratory chemical and is widely us ed in indus try, agric u lture, medical
practice, and organic s ynthes is res earch. It is a common bactericide, pes ticide, and indus tria l n it ro g e n gas
g e n erator, and known to be highly mutagenic in s everal organis ms , including plants and animals (Rines , 1985;
Velemins ky and A nglis , 1987; Raicu and M ixich , 1992). It has been reported that NaN 3 affects plant
phys iology and decreas e cyanide res is tant res piration in tob a c c o callus (W en and Liang, 1995). The
mutagenicity of this chemical is mediated throug h t h e production of an organic metabolite of azide compound
(Owais and Kleinhofs , 1988). W ith reference to the mutagenic effec t o f N a N 3 at different concentrations in
barley, s ome res ults are contradictory, probably due to the different treatmen t c o n d it io n s (pres oaking, pH,
temperature, time of expos ure, etc.) and to the different varieties us ed (Ilbas et a l . 2005). Sodium azide has
been us ed in a n u mber of crops for s everal biotic and abiotic s tres s es s uch as Zea mays res is tant agains t
pathogen Striga (Kiruki et al. 2006), Musa spp. A A A res is tant agains t Fusarium oxysporum f. s p. cubens e
(Bhagwat and Duncan, 1988), barley res is tan t agains t Mildew dis eas e (M olina-Cano et al. 2003), Saccharum
officinarum res is tant agains t red ro t d is eas e (A li et al. 2007), Araches hypogea (M ondal et al. 2007), Lactuca
sativa res is tant agains t down mildew dis eas e (Okubara et al. 1994), Glycine m a x for enhanced fatty acid
content (Hammond and Fehr, 1983b), Triticum aestivum (durum wheat) for s alt tole rance (A gata et al. 2001),
Oryza sativa for reduced amylas e content (Jeng et al. 2003), Oryza sativa for enhanced yield (J e n g e t al.
2006), Halianthus annuus for enhanced s tearic acid content (Skoric et al. 2008), Halianthus annuus for reduced
triacylglycerol content (Venegas -Caleron et al. 2008), Oryza sativa for s ilicon deficient (Naka t a e t a l . 2008),
Hordeum vulgare for reduced phytic acid cont e n t (O liver et al. 2009), Oryza sativa for enhanced amylas e
content (Suzuki et al. 2008) and Zea mays for drought t o le ra n c e (He et al. 2009) res pectively. The s odium
azide mutagenicity was performed on E. sativa in green hous e experiment, and 3 mM co ncentration s howed
revers ible inhibitory effect on growth and yield t ra it s a ft er 60 days of s owing (A l-Qurainy et al. 2009). In the
light of above literature, in the pres ent s tudy, the mutagenic effect o f various concentrations of NaN3 on s eed
germination and s eedling growth were s tudied on E. sativa after a time interval in petriplate.

MATERIALS AND METHODS

The s eed of E. sativa was purchas ed from a local market of Riyadh, and the experiment was conducted
at the Department of Botany and M icrobiology, King Saud Univers ity, Riyadh, Kingdom of Sa u d i A rabia. The
N a N 3 w a s dis s olved in water and s tock s olution made of 1.5 M concentration. Further, it was diluted with
0.1M s odium pho s p h a t e b u ffe r of pH 3.2, and dilution was made of various concentrations ranged from 1 to
5 mM . It was als o diluted in dis tilled water, and dilution was made of various concentrations ranged from 1
to 5 mM , and further us ed for s eed treatment for various time intervals .
The s eeds were chos en after pas s ing through s ieve of s ize 1.5 x 2 mm. They w ere s oaked into autoclaved
dis tilled water for 12 h with agitation on s haker at room temperature. A fter s oaking into water, they were
was hed with autoclaved dis tilled water for five times to remove brown colour appeared from s eeds in water.
A ft e r w a s h in g, 50 s eeds were kept in various concentrations of NaN 3 s olution for 30 min, 60 min, 120 min
and 180 min with agitation on s h a ke r a t ro o m temperature. A fter NaN3 treatment, s eeds were was hed with
a utoclaved dis tilled water for five times to remove excess NaN3 , and thereafter, each group of s eeds (50 e a c h
treatment) were trans ferred to wet W hatmann paper in p e t ri d is h es at 21 ºC for the inves tigation of the
mutagenic effects of NaN3 . The % germination and s eed lin g g ro wth were inves tigated after 9 days of s owing
in all treated and untreated s eeds .

Statistical Analysis:
Statis tical s ignificance was evaluated with one-way A NOVA analys is followed by D u n n e t t ’s multiple
comparis on tes t (c o mparing s eedling developed from treated s eeds with NaN3 to untreated s eedling, and als o
among s eedlings of treated s eeds ).

RES ULTS AND DIS CUS S ION

Sodium azide is highly s o lu b le in w a t er, but fewer number of hydrozoic ions are produced in water, and
at low pH the quantities of NaN3 dis s ociated to hydrozoic acid which is theoretically many times greater (at
pH 3 t h e re is a pproximately 19 times more hydrozoic acid than at pH 6, for the s ame concentration of NaN3 ),
and tha t w o u ld be the condition for better penetration through the cell membrane and create mutations in the

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genome of a cell (Nilan e t a l. 1973; Kleinhofs et al. 1974). The pH value of s oaking s olution affects the
efficiency of mutation with NaN3 . W e perfo rme d o u r e xperiments in two dilution s ys tems , water as well as
phos phate buffer of pH 3.2. The little mutagenic effe c t o f N a N 3 or negligible effect was obs erved in water
us ed as dilution s ys tem (data not s hown). W hen it was diluted in pho s p h a t e b u ffe r o f pH 3.2, it had s trong
mutagenic effect on % germination, radicle and coleoptile lengths res pectively.
In our experiment, 180 min treatment duration was very effective and at this treatment duration %
germination was found to be 51.11(1 mM ), 28.89 (2 mM ), 22.32 (3 mM ), 6.66 (4 mM ) and 4.04 (5 mM )
res pectively. The s eeds t reated for 120 min with s ame concentration as 180 min, % germination was higher
at 120 min of time interval th a n t hat of 180 min of time intervals , and it was found to be 62.21(1mM ), 42.02
(2 mM ), 26.56 (3 mM ), 17.78 (4 mM ) and 11.12 (5 mM ) re s p e c t iv e ly . S imilarly, treatment for 60 min of time
interval with NaN3 , the % germination was found higher than 180 min and 120 min o f t ime interval, and it
was found 51.11(1 mM ), 45.44 (2 mM ), 40 (3 mM ), 22.78 (4 mM ) and 13.33 (5 mM ) res pectively. The %
germin ation was found maximum when s eeds were treated for 30 min of time interval. Our res ult was line
with the res ult of A d mu a n d A liy u (A damu and A liyu, 2007) who performed your experiment on tomato, and
treatment of NaN3 was very effective in in d u c in g mutations and affects the % germination, root length,
s eedling height, s eedling s urvival, number of branches per plant, and yield per plant. The various concentration
of NaN3 als o affected the s e e d ling s urvival, and reduction in s eedling s urvival is attributed to the cytogenetic
damage and phys iological dis turbances (Sato and Gaul, 1967; Natrajan and S h iv s h a nkar, 1965). The greater
s ens itivity at higher mutagenic dos e has been attributed to various fact o rs s u c h as changes in the metabolic
a c t ivity of the cells , inhibitory effects of mutagens (M aherchandari, 1975) and to dis turbance of b a la n c e
between promoter and inh ibitors of growth regulators (Kris hna et al. 1984). The % s eed germination was
decreas ed at all s tudied concentrations of NaN3 at various time intervals as compared to untreated s eeds , which
had 85 % of s eed germination in all exp e rime n t a l g ro u p s . The invers e relation was found among the various
concentrations of NaN3 for various time intervals and percent s eed germination (Table 1). The reduction in s eed
germination in mutagenic treatments had been explained due to delayed or in h ib it io n o f phys iological and
biological proces s es neces s ary for s eed germination which include enzyme activity (Ch ris peeds and Varner,
1976), hormonal imbalance (39) and inhibition of mitotic proces s (A nanthas wamy et al. 1971). The inhibitory
effect of NaN3 on germination could be azide anions which are s trong inhibitors of cytochrome oxida s e, which
in turn inhibits oxidative phos phorylation (Kleinhofs et al. 1978). In addition, it is a poten t in h ib itor of the
proton pump and alters the mitochondrial membrane potential (Zh a n g , 2000). Thes e effects together may
hamper A TP bios ynthes is res ulting in decreas ed a v a ilability of A TP which may s low the germination rate and
reduce the germination percentage. Cheng and Gao (Cheng and Gao, 1988) treated barley s eeds w it h N a N 3
and s ignificant reduction was found in the % germination. Furthermo re, the effect of NaN3 was meas ured after
5-7 days from s owing, when the length of the firs t leaf had not reached its maximu m, re n d e rin g it impos s ible
to dis tinguis h between delay in germination, and a real length reduction (Gaul, 1970; Kon za k e t a l . 1975). A s
the NaN3 concent ration increas ed from 1 mM to 5 mM , there was delay in s eed germination, and s eeds treated
at 5 mM concentration of NaN3 fo r v a rio u s time intervals (30, 60, 120 and 180 min) s howed s trong mutagenic
effe c t on germination, and it was found to be 55.54%, 13.30%, 11.12% and 4.04% res pectively. The %
germination, radicle and coleoptile lengths were profoundly affected, when s eed treated with NaN3 , diluted into
phos phate buffer of pH 3.2. The radicle and coleoptile length were s trongly affected by NaN 3 treatment and
as the dos e of NaN3 increas ed, the radicle and coleoptile length were decreas ed, but more effect was obs erved
on radicle length (Fig 1). A mong various time in t e rv a ls , the s eeds treated for 180 min s howed s trong
mu t a g e n ic affect on radicle and coleoptile lengths . The s eeds treated with various concentrations of NaN3 a t
3 mM , 4 mM and 5 mM for 180 min of time intervals s how ed high mutagenic effect on radicle length (cm),
and it was found to b e 2.22 ± 0.08, 1.40 ± 0.08 and 0.80 ± 0.35 res pectively (Table 3, Fig 2). Similarly, the
length of coleoptile (cm) at above concentration was found to be 1.56 ± 0.12, 1.06 ± 0.09 and 0.80 ± 0.08
at various concentrations of NaN3 including 3mM , 4mM and 5mM res pect iv e ly (T a b le 2, Fig 3). The
concentratio n s 1 mM and 2 mM of NaN3 for various time intervals s howed les s mutagenic effect on radicle
and coleoptiles le n g t h as compared to 3 mM , 4 mM and 5 mM (Fig 1). Our res ults s howed that the high dos e
of NaN3 (5 mM ) treatment at various time intervals s h o w ed high mutagenic effect on E. sativa and it is
mentioned in literature that treatments with NaN 3 at various concentrations , under the s ame conditions , produce
a delay in the initiation of plant g ro w t h , as can be obs erved and mentioned by Pears on et al. (1974, 1975).
Kleinhofs et al. (1978b) s ugges ted that 0.003 M NaN3 dos e increas ed mutations in pea. The higher dos e of
NaN3 als o caus ed dis turbance in g e n e t ical and phys iological activities leading to the death of the cells . Prina
and Favret (1983) us ed 0.001 and 0.005 M dos es of NaN3 on barley, but could not detect any phys iological
changes o n t h e s hoot development. In conclus ion, NaN3 is a s trong mutagen, and affected s eed germination,
radicle and coleoptile lengths of E. sativa, and thus it s hould be us ed further on t h is s p e c ie s t o improve its
agronomic traits and als o produce res is tance to them agains t biotic and abiotic s tres s by creating mutation.

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Table 1: M u t agenic effect of various concentration of NaN3 on percent seed germination at various time intervals (after 9 d ay s o f
sowing). Higher doses of NaN3 produced profound effects on germination for long time intervals.
T reatment T ime intervals (min)
--------------------------------------------------------------------------------------------------------------------
30 60 120 180
Control 85 85 85 85
NaN3 (1 mM) 71.61 55.01 62.21 51.11
NaN3 (2 mM) 64.54 45.04 42.02 28.89
NaN3 (3 mM) 60.00 40 26.56 22.32
NaN3 (4 mM) 57.37 22.78 17.78 6.66
NaN3 (5 mM) 55.54 13.30 11.12 4.04
Values are mean ± SD for three replicates in each group.
a p< 0.01 when compared with control b p< 0.001 when compared with control NS (non significant)

T a b l e 2 : M u tagenic effect of NaN3 on coleoptile length at various time intervals (after 9 days of sowing) done in petriplate experi m en t .
T reatment T ime intervals (min)
--------------------------------------------------------------------------------------------------------------------
30 60 120 180
Control 3.40±0.15 3.20±0.15 3.16±0.12 3.06±0.16
NaN3 (1mM) 3.33±0.09ns 3.06±0.04ns 2.80±0.08ns 2.16±0.24a
NaN3 (2mM) 3.08±0.04ns 3.03±0.04ns 2.43±0.36ns 1.86±0.18a
NaN3 (3mM) 3.01±0.06ns 2.90±0.08ns 1.86±0.09a 1.56±0.12b
NaN3 (4mM) 2.90±0.08ns 2.43±0.12a 1.30±0.08a 1.06±0.09b
NaN3 (5mM) 2.70±0.08a 2.03±0.12b 1.00±0.08a 0.80±0.08b
Values are mean ± SD for three replicates in each group.
a p< 0.01 when compared with control b p< 0.001 when compared with control NS (non significant

Table 3: Mutagenic effect of NaN3 on radicle length at various time intervals (after 9 days of sowing) done in petriplate experiment.
T reatment T ime intervals (min)
--------------------------------------------------------------------------------------------------------------------
30 60 120 180
Control 5.10±0.28 4.63±0.33 5.03±0.04 4.80±0.24
NaN3 (1mM) 5.03±0.04NS 4.63±0.12NS 4.63±0.12NS 4.06±0.12NS
NaN3 (2mM) 4.73±0.09NS 3.56±0.09NS 3.52±0.22a 3.31±0.08a
NaN3 (3mM) 3.80±0.08NS 2.73±0.12a 2.58±0.14a 2.22±0.08b
NaN3 (4mM) 3.63±0.12a 1.80±0.08b 1.58±0.25a 1.40±0.08b
NaN3 (5mM) 3.40±0.16a 1.56±0.04b 0.93±0.04a 0.80±0.35b
Values are mean ± SD for three replicates in each group.
a p< 0.01 when compared with control b p< 0.001 when compared with control NS (non significant)

Fig. 1: Effe c t o f v arious concentration of NaN3 on coleoptile and radicle length for 180 min of time interva l,
and photographs were taken after 9 days of s owing in petriplate experiment.
Control (0 mM ) NaN3 1 mM NaN3 treated s eeds
2 mM NaN3 treated s eeds 3 mM NaN3 treated s eeds
4 mM NaN3 treated s eeds 5 mM NaN3 treated s eeds

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Fig. 2: M utagenic effect of various concentration of NaN 3 on co le o p tile length at various time intervals , and
all treatments were co mpared to control. Res ults s hows that treatment with NaN3 decreas ed coleoptile
length. Data are mean ± S D for three replicate done petriplate. Statis tical s ignificance was determined
by A NOVA (Dunnett’s multiple comparis on tes t).
Values are mean ± S.D for three replicates in each group
a p< 0.01, when compared with control
b p< 0.001, when compared with control
ns - Not s ignificant

Fig. 3: M utagenic effect of various concentration of NaN 3 on radicle length at various time interv a ls , and all
treatments w e re c o mp a red to control. Res ults s hows that treatment with s NaN3 decreas ed radicle
length. Data are mean ± SD for three replicate done petrip la t e. Statis tical s ignificance was determined
by A NOVA (Dunnett’s multiple comparis on tes t).
Values are mean ± S.D for three replicates in each group
a p< 0.01, when compared with control
b p< 0.001, when compared with control
ns - Not s ignificant

ACKNOWLEDGEMENT

The auth o r is thankful to the Res earch Centre, College of Science, King Saud Univers ity, Riyadh,
Kingdom of Saudi A rabia, for providing funds to carry out this res earch program.

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