1

Gibberellic acid and Cold Effect on Protein Variation

in Ferula gummosa Boiss. seeds
Shirin Haddad Kaveh*, Zeinab Pasban Vatan and Franoise Bernard
Plant Physiology and Biotechnology Laboratory, Faculty of Biological Sciences University of Shahid
Beheshti ,Tehran, Iran

ABSTRACT
Seeds of Galbanum (Ferula gummosa Boiss.) are characterized by a very low rate of germination in the
laboratory condition due to the difficulties to find efficient breaking factors of the complex dormancy of
these seeds. To some extent gibberellic acid (GA3) and cold temperatures can contribute to the removal of
dormancy. In this study the effects of gibberellic acid pretreatments (0, 500, 1000, 1500 mM) and different
temperatures (-20C, 4C, 8C) given during seeds soaking step were measured on changes in
electrophoretic patterns of proteins of different treated samples. The seeds pre-treated with 500 mM GA 3 or
4C germinated with a germination rate of 22% and 8% respectively. Lots of seeds, treated by other
temperature conditions, which were not germinated, have an electrophoretic profile of proteins mainly
characterized by the absence of three polypeptide bands. These bands are present in the protein fraction of
seeds treated with GA3 and 4 C even if the seeds did not have germinated. A 23kDa polypeptide not well
present in GA3 or 4C treated germinated and non-germinated seeds, was well presented in recalcitrant seeds.
The comparison with the standard profile of alpha- amylase shows that two of these polypeptide bands
correspond to this enzyme. The heterogeneity of F.gummosa response to dormancy breaking treatment was
accompanied by changes in the levels of some peptides of interest in order to study further in the future.
These results also highlight the role of GA3 and coldness on the synthesis of alpha-amylase involved in the
metabolic activation for seeds germination of Ferula gummosa.
Key words: dormancy galbanum - alpha-amylase - germination.
Abbreviations: GA3 Gibberellic Acid; ABA- Abscisic acid
Haddade Kaveh, Sh* , Pasban Vatan ,Z and Bernard , F (2010) Gibberellic acid and Cold Effect on Protein
Variation in Ferula gummosa Boiss. seeds .Iranian J of Plant Physiology, 1(1): 1- 6 .

INTRODUCTION
Ferula gummosa Boiss, a monocarpic plant from
Umbelliferae family, is prized for its oleogum called
galbanum, a mixture of essential oil and resin that is
produced in the tuber of this perennial plant.
For a long time galbanum oil was used for different
medicinal and spiritual purposes and, as written in
the book of Exodus [30:34], it was the favourite oil
of Moses. Two thousand years ago, in Egypt,
*Corresponding author: shkaveh@yahoo.com
Tel : +98-912-7040115
Received: July, 2010
Accepted: September, 2010

galbanum by Bess wax and bitumen were used in

the linen of mummy wrappings (Benson et al,
1978). It was used as antiseptic, antispasmodic, anti
inflammatory and antitoxic in the past (Zargari,
1991). Today, F. gummosa is recognized for its
antibacterial (Eftekhar et al, 2004) and health
promoting properties. Many essential oil compounds
of galbanum are very important for medicinal
(Sadraei et al, 2001; Sayyah et al, 2002 ; Ramezani
et al, 2001) industrial and perfumery uses.The
major habitat of Ferula gummosa is high altitude
mountains of Iran(Ghahreman, 1986) and this
country is the most important exporter of galbanum

Iranian Journal of Plant Physiology, Vol (1) , No (1)

gum. Cultivation of galbanum has not been

achieved and intensive harvesting from natural
habitat in different zones is a real threat for its life.
Ferula seeds are characterized by severe and
multiple dormancy (Rahnama-Ghahfarokhi et al,
2007, Keshtkar et al, 2008) that prevent good
recovery of germination in laboratory Different
techniques have been tested for breaking dormancy
of Ferula gummosa seeds and among them coldness
and GA3 treatments have been showed to partially
improve Ferula seed germination (Nadjafi et al,
2006 ; Rahnama-Ghahfarokhi et al, 2007; Keshtkar
et al, 2008). However no reports have been done
about the metabolic changes due to the effects of
GA3 and cold treatment in Ferula seeds. In this
study, the effect of coldness and GA3 treatments on
changes of protein electrophoretic profiles was
examined in galbanum seeds.
.

MATERIAL AND METHOD

Seed Collection
Galbanum seeds were collected from Firouzkouh
Mountains (Iran) in September 2008 and they were
kept in 4C until used for the experiment.
Germination in different treatments
Firstly the disinfection treatment was carried out
using 70% ethanol (V/V) for 30 seconds. Then the
seeds were rinsed with 10% H2O2 (V/V) for 10
minutes andthentransferred to1%(V/V) hypochlorite.
Three groups of 100 disinfected seeds which
initially rinsed by distilled water, were finally
distributed in sterile Petri dishes. One of the series
was pretreated with GA3 (0, 500, 1000, 1500 mM)
for 48 hours and then placed at 4C. The second
group was kept at -20 C for 2 weeks and then
transferred to 4 C, and the third group was stored
at 4C for 2 weeks and then transferred to 8 C.
Petri dishes were sealed using parafilm.
Protein Analysis
An amount of 0.3 g of seed was used for
extraction of proteins according to Wang et al.
(2006) method, suitable for tissues with high level
of phenolics. Protein measurement was conducted
2, 4, and 6 months after seeds harvesting. Total
proteinanalysis was performed according to Bradford
(1976)test andelectrophoresis for qualitative
measurements of proteins was performed by SDSPAGE. Alpha-Amylase marker (Merck) was also
used as a standard.

Statistical Analysis
All data in this study were statistically analyzed
using LSD test and analysis of variance (ANOVA)
by SPSS 11 software.

RESULTS
Ferula gummosa Seed Germination
The results of germination are shown in table 1.
GA3 treatment stimulated F. gummosa seeds
germination. In our experiment 48 h soaking by 500
mM GA3 show the best improvement for the
germination percentage. This treatment was
effective in increasing germination up to 22%.
Higher concentration of GA3 did not show
significant differences in germination process of
galbanum seeds. F. gummosa seeds responded also
to 4C cold treatment but in a less amount.These
results showed a high heterogeneity in seeds
population as a high percentage of seeds did not
respond to the GA3 and 4C cold treatment ( see
table 1).The percentage of germination in other
treatments for temperature was very low nearly
zero.
Qualitative Protein Analysis
Non germinated and germinated treated seeds
were used to compare the electrophoretic protein
profiles of these two categories of seeds that
showed different sensitivity to dormancy-breaking
treatment. Figure I shows protein profiles of seeds,
treated by GA3 and different temperature treatments,
that have not germinated. One may note two types
of profiles. The first type for the seeds treated by
GA3 or treated by 4C. The second type, for the
seeds treated by other temperature treatments that
were inefficient for germination, the later one is
differed from the first one principally by the lack of
three polypeptides bands of about 11 kDa, 42 kda
and 57 kDa. In this category of seeds the
polypeptidic band of 23 kDa was more obvious than
in the first type of non germinated seeds treated by
1000mM GA3. Although germination was not
observed, the treatments affected protein
metabolism. If we compare the protein profiles of
seeds efficiently treated by GA3 or 4C for breaking
dormancy,onemay note that the 11 kDa polypeptide
in these profiles is practically absent compared to
the second type of non germinating seeds. In
germinated seeds the 42 kDa polypeptide was
present with an intensity dependant to the duration
of culture (Figure II). It is noteworthy that 23 kDa
band appeared very weakly and is practically absent

Gibberellic acid and cold effect of seed of Ferula gummosa

in germinated seeds treated by 1000mM GA3 and

4C. Figure II showed also that, in germinated
seeds, two new bands emerged. Interestingly the
main change between non germinated seeds and
germinated seeds bring two bands that corresponded
with 28 and 55 kda polypeptides of -amylase
marker.

DISCUSSION
Although the phenomenon of seed dormancy has
been a subject of many investigations for a very
long time, it is not yet fully identified. One reason
perhaps would be that dormancy is expressed and
released in different ways depending on the species.
The mechanisms of dormancy may settle in the seed
coat (coat-enhanced dormancy) or in the embryo
(embryo dormancy). F. gummosa seeds are
affected by two types of dormancy which
complicates studies on the dormancy of these seeds.
Another difficulty in the study of dormancy is that
the process does not appear synchronously in a
population of seeds (Bewley, 1997), and this is
particularly the case in the population of F.
gummosa as we have noted in our study as well.
The treated samples responded to gibberellic acid
and coldness are in agreement with other authors
who worked on this subject (Keshtkar et al, 2008;
Nadjafi et al, 2006) but with a relatively low
germination percentage (22% for maximum
response to 500 mM GA3 and 8% in response to
4C cold) meaning an asynchronisation in the
response to treatment of this population of seeds. If
gibberellic acid can counteract with the inhibitory
effect of abscisic acid (ABA) on germination, GA 3
is first an important factor in the initiation and
maintenance of germination (Bewley and Black,
1982; Bewley and Black, 1994). Also the sensibility
to GA3 may be a key factor in the response of seeds
in relation to the presence of receptors for dormancy
-breaking agents (Hilhorst,1993; Vleeshouwers et al,
1995).We have noted that under the effect of GA 3
and coldness or in a lesser extent under the effect of
cold treatment at 4C alone, seed protein
metabolism was altered but in different ways and
this response may depend on difference in

sensitivity of seeds to gibberellic acid. This could

explain the asynchronicity in the germination of
seeds of the study population. Specifically, under
GA3 and 4C treatment, a 42 kDa polypeptide
which was present in large amount in non
germinated seeds was also found in germinated
seeds but innless and variable amounts directly
related to the period of seed incubation. Seeds
treated by temperature treatments that are inefficient
in dormancy release and have not promoted seed
germination, did not show this poplypeptide in their
protein electrophoretic profile. On the contrary, the
high presence of the 23 kDa protein in this last
category of non germinating seeds suggests that this
peptide could play a role in dormancy. We can
therefore assume that 4C cold treatment or more
intensively GA3 and coldness act on the synthesis
pathway of 42 kDa and 23 kDa polypeptides and
these results changes may be implicated in the
release of dormancy. The isolation and
characterization of these polypeptides may help to a
better knowledge about the breaking dormancy
process. The seeds that germinated under the effect
of GA3 also showed de novo synthesis of
polypeptides that could correspond to some
isozymes of alpha-amylase by comparison with
alpha-amylase standard (Figure II). During
germination GA3 control of the activity of alphaamylase has well been studied in monocotyledons
plants (Jacobsen and Higgins, 1982) but less work
has reported its effect on germination of
dicotyledons species. It is very probable that alphaamylase isozymes play a key role in the
mobilization of carbohydrates for a normal growth
of F.gummosa seedlings. In a further study we can
isolate the F. gummosa alpha-amylase isozymes and
measure specific changes in the activity of these
isozymes under GA3 treatment. In our laboratory
we also have isolated an embryogenic cell line of F.
gummosa (Bernard et al, 2007). Somatic embryos
that proliferate extensively in vitro show no
dormancy and germinate very quickly. Comparison
of protein metabolism between these somatic
embryos and seeds embryos will help us advance
the knowledge of seed dormancy of Ferula
gummosa.

Iranian Journal of Plant Physiology, Vol (1) , No (1)

Table 1: Ferula gummosa seed germination during 6 weeks of culture (100 seeds per treatment)

Soaking step treatment

temperature
treatment
during
germination
step

% of germination along the duration of

culture in week
1

4 C

500

4C

10

15

19

22

1000

4C

13

17

19

1500

4 C

11

16

18

-20C

4 C

8C

8C

24C

24C

48 h
GA3 soaking
treatment
at 4C (mM)

2 weeks soaking
treatment at different
temperature

Gibberellic acid and cold effect of seed of Ferula gummosa

Figure I: Electrophoretic profile of seed proteins of Ferula gummosa in not germinated seeds: alphaamylase marker (A); seeds pretreated with 500mM GA 3 after two (1), four (2) and six weeks (3) of culture at
4C; seeds pretreated with 1000mM GA3 after two (4), four(5) and six weeks (6) of culture at 4C; seeds
pretreated at 4C after two (7), four (8) and six weeks (9) of culture at 4C; seeds pretreated at -20C after
six weeks of culture at 4C (10); seeds pretreated at 8C after six weeks of culture (11); seeds pretreated at
24C after two (12) , four (13) and six weeks (14) of culture at 24C.

Figure II. Electrophoretic profile of proteins in germinated (1, 2, 3, 4, 5, 6, 7, 8, 9)and not germinated (10,
11, 12, 13, 14)seeds of Ferula gummosa: alpha-amylase marker (A); seeds pretreated with 500mM GA 3 after
two (1), four (2) and six weeks (3) of culture at 4C; seeds pretreated with 1000mM GA 3 after two (4),
four(5) and six weeks (6) of culture at 4C; seeds pretreated at 4C after two (7), four (8) and six weeks (9)
of culture at 4C; seeds pretreated at -20C after six weeks of culture at 4C (10); seeds pretreated at 8C
after six weeks of culture (11); seeds pretreated at 24C after two (12), four (13) and six weeks (14) of
culture at 24C. (arrows show corresponding bands with 28 and 55 kDa -amylase marker peptides).

Iranian Journal of Plant Physiology, Vol (1) , No (1)

REFERENCES
Benson, G.G., Hemingway, S.R. and Leach, F.N
(1978) Composition of wrappins of an ancient
Egyptian mummy, J. Pharmacy Pharmacol. ,30:
123-129.
Bernard, F., Shaker, H., Javadi-Khatab, L.,
Shafiei-Darabi, A., and Sheidai M (2007)
Ferula gummosa Boiss. embryogenic culture
and karyological changes, Pak. J. Biol.Sci.10
(12) : 1977 - 1983.
Bewley, J.D (1997) Seed germination and
dormancy, Plant cell, 9:1055-1066.
Bewley, J.D., and Black, M (1982) Physiology and
Biochemistry of Seeds in Relation to
Germination. 2. Viability, Dormancy and
Environmental Control Berlin: Springer-Verlag.
Bewley, J.D., and Black, M (1994) Seeds:
Physiology of Development and Germination
New York: Plenum Press.
Bradford, M.M (1976) A rapid and sensitive
method of quantity of microgram quantities of
protein utilizing the principle of protein dye
binding Analytical Biochemistry, 72 : 248-254.
Eftekhar, F., Yousefzadi, M., and Borhani, K
(2004) Antibacterial activity of the essential
oil from Ferula gummosa seed, Fitoterapia, 75:
758759.
Ghahreman, A (1986) Flora of Iran, Tehran: RIFR
Publication.
Hilhorst, H.W.M (1993). New aspects of seed
dormancy. In Fourth lnternational Workshop on
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Biology, Cme, D., and Corbineau, F., eds,
Paris: ASFIS.
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Characterization of the -amylases synthesized by
aleurone layers of Himalaya barley in response
to gibberellic acid, Plant Physiol. 70: 1647-1653.
Keshtkar, H.R., Azarnivand, H., Etemad, V., and
Moosavi, S.S (2008) Seed dormancy-breaking

and germination requirements of Ferula ovina

and Ferula gummosa, Desert, 13: 45-51.
Nadjafi, F., Bannayana, M., Tabrizi, L., Rastgoo,
M (2006) Seed germination and dormancy
breaking techniques for Ferula gummosa and
Teurium polium, Journal of arid environment,
64: 542-547.
Rahnama-Ghahfarokhi, A., and TavakkolAfshari R(2007) Methods for dormancy breaking
and germination of galbanum seeds (Ferula
gummosa), Asian J. Plant Sci. 6: 611-616.
Ramezani, M., Hosseinzadeh, H., and Mojtahedi,
K (2001) Effect of Ferula gummosa Boiss.
fractions on morphine dependence in mice, J.
Ethnopharmacol , 77: 71-75.
Sadraei, H., Asghari, G.R., Hajhashemi, V.,
Kolagar,A., and Ebrahimi, M (2001)
Spasmolytic activity of essential oil and various
extracts of Ferula gummosa Boiss. on ileum
contractions, Phytomedicine, 8(5):370-376.
Sayyah, M., Mandgary, A., and Kamalinejad, M
(2002) Evaluation of the anticonvulsant activity
of the seed acetone extract of Ferula gummosa
Boiss against seizures induced by
pentylenetetrazole and electroconvulsive shock
in mice, J. Ethnopharmacol. 82 :105-109.
Vleeshouwers, L.M., Bouwmeester, H.J., and
Karssen, C.M (1995) Redefining seed
dormancy: An attempt to integrate physiology
and ecology. J. Ecol, 83: 1031-1037.
Wang, W., Vignani, R., Scali , M., and Cresti, M
(2006) A universal and rapid protocol for
protein extraction from recalcitrant plant tissues
for proteomic analysis, Electrophoresis, 27:2782
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Zargari, A (1991) Medicinal Plants Tehran: Tehran
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The Study Of Steroid Hormone Effects on the Rate of Growth and

Gruit Body Formation in Pleurotus florida (Fr.)Sing

Sara Saadatmand *, Hamid Fahimy, Nasrin Sartipnia

Islamic azad University, Science and research branch,Tehran, Iran.

ABSTRACT
In this study we examined effects of the different concentrations of 17- estradiol and progesterone (0
(control), 1.5, 3.02, 6.05 M) on the growth rate and fruit body formation of Pleurotus florida. We cultured
Pleurotus florida in Potato Dextrose Agar (PDA) medium for 20 days and measured the diameters of
colonies after the 6th and 7th day of inoculation. Then protein content was measured by Lowry Method and
protein profile determined by SDS-Page. The results showed that the estroidal (estradiol) treatments
increased the rate of growth in comparison with the control. Also the results showed that in 6.05 M
progesterone treatment, the colonys diameters were higher than the other treatments. In 1.5 M estradiol,
fruit body formation was stimulated on the 12th day after treatment. In this treatment (1.5 M estradiol) we
showed that protein content was higher than the other samples. In the different hormonal treatments we
showed vertical growth besides horizontal growth. The Gel electrophoresis of proteins showed that some
polypeptide bands with low molecular weight were absent in the different steroid treatment.
Keywords: estradiol, Pleurotus florida, progesterone, SDS-PAGE, pin head.
Saadatmand Sara*, Fahimy H., Sartipnia ,N. (2010) The study of steroid hormone effects on the rate of
growth and fruit body formation in Pleurotus florida (Fr.)Sing.Iranian J of Plant Physiology ,1(1): 7 - 12 .

INTRODUCTION
Pleurotus florida is an excellent edible mushroom,
hence P. florida is cultivated on a commercial scale
in many parts of the world, including Iran. This
mushroom isa nutritionally functional food with
valuable therapeutic use. The best known therapeutic
agents that it is stated to be of potential use for
correcting hypercholesterolemia are levostatin and
its analogues. Pleurotus species are reported to be
the best known source of this medicament (Nayana
and Janardhanan 2000).These species are commonly
called Oyster mushrooms. There are about 40
species of this mushroom. They enjoy worldwide
distribution, both in temperate and tropical parts of
the world. Oyster mushrooms now are ranked in the
second among the important cultivated mushrooms
in the world (Nayana and Janardhanan 2000).
*Corresponding author: s_saadatmand@srbiau.ac.ir
Tel : +9821-44865323
received: July, 2010
Accepted: Augnst, 2010

Wolter et al. (1997) proposed that P. florida is

suitable for bioremediation of contaminated soils
because of its ability to degrade highly condensed
polycyclic aromatic hydrocarbons (PAHs) and its
high tolerance of these substrates. Nutritionally, the
fruiting body of the mushroom has been found to
contain vitamins B1 (thiamin), B2 (riboflavin), B5
(niacin), B6 (pyridoxine and B7 (biotin) (Solomko
and Eliseeva 1988) and it is also a potential source
ofligninandphenol degrading enzymes (Fountoulakis
et al. 2002). Knowledge of the cellular processes
leading to the initiation of fruit body development is
lacking in several edible mushrooms, including P.
ostreatus. Therefore, the identification of the genes
and proteins involved in fruiting, as well as
studying theeffectsof environmental and biochemical
treatments on fruit body formation are extremely
important biotechnologically and commercially.
These findings can be used to control fruit body
initiation, which is the pivotal step in the production
of mushrooms.
Inthis study effects of the different concentrations
of 17- estradiol and progesterone (0 (control), 1.5,

3.02, 6.05 M) were examined on the growth rate

and fruit body formation of Pleurotus florida .

MATERIALS AND METHODS

Fungal cultivation and incubation
Pure mycelial culture of P. florida was obtained by
tissue culture method (Jonathan and Fasidi, 2003).
The mycelial culture was maintained on Potato
Dextrose Agar (PDA) in the dark at 27C and 70%
relative humidity. After the colonies reach a
diameter of 25 to 35 mm, the plates are inverted and
incubated at 12 to 15C .In the Petri plate, two disc
of this culture were inserted. After 2-3 days, culture
was used for inoculation. Wells (6mm diameter)
were punched in the agar and filled with 30 l of
hormone added into well. Triplicates of each plate
have been performed. The plates were incubated at
37C for 24 h. The growth rate was assessed by
measuring the diameter of the area in which
represents the mycelial growth around the well. The
average of four replicates for each treatment was
calculated.
Determination of protein
The protein content of culture broth was determined
by Bradford method with bovine serum albumin as
standard (Bradford ,1976).
Statistical analysis
Mean values of biomass yield were analyzed by
one way Analysis of variance (ANOVA) and tests of
significance difference were determined by
Duncans multiple range tests (Snedecor and
Cochran , 1987).

RESULTS
The results showed that in the all hormonal
treatments, the mycelial growth increased in
compare with control (Fig1). The best mycelial
growth was in 1.5 M progesterone in the 6th day
after inoculation and 3.02 M progesterone in the
7th days after inoculation at P< 0.05. As well as, the
progesterone treatments were the best treatments for
mycelial growth in compare with the esteradiol
treatments (Fig2).
The results shown in Figure 3 indicates that P.
florida produced pin heads in 1.5 and 3.02 M
esteradiol and 6.05 M progesterone.
Fig 4 shows that protein content increased in all
treatments and these results were significantly

different from each other and control sample at P<

0.05, except of 1.5 M estradiol. Generally, it was
observedthat the highest concentration progesterone
(6.05 M) increased the protein content of P.
florida higher than the other treatments and its not
significantly different from protein content of 1.5
m estradiol and 3.02 M progesterone treatments.
The results of protein profile by SDS-Page showed
that in 1.5M estradiol, 1.5 and 3.02 M,
progesterone was a 28 KD protein band which is
not present in control and the other treatments (Fig
5). In 1.5 M progesterone treatment showed the
most clear polypeptide band.

DICUSSION
Based on the results of this study, it can be
concluded P.florida has esteroidal receptors and 17
-estradiol and progesterone treatments has effects
of fruit body formation. The identification of the
genes and proteins involved in fruiting, as well as
studying theeffectsof environmental and biochemical
treatments on fruit body formation are extremely
important biotechnologicallyandcommercially.
Hormones control andcoordinatecomplex
physiological and developmental processes in
plants, animals and fungi, such as growth,
differentiation reproduction and homeostasis. Plant
steroids and terpenes are widespread and ancient,
and function as insect feeding deterrents in many
cases (Chory , 1999; Pare and Tomlinson ,1999).
Steroid hormones, estrogen and progesterone,
profoundly influence the development and function
of the female reproductive system. The hormonebound receptor interacts with specific genes in the
responsive tissue and regulates their expression.
The white-rot fungus Pleurotus ostreatus has so far
been found to metabolize polycyclic aromatic
hydrocarbons (Bezalel et al. 1996) as well as to
oxidize androgens and estrogens (Lanisnik et
al.1992). The Pleurotus osteratus 17b-HSD enzyme
preparation was found to have a broad substrate
specificity catalyzing efficiently the oxidation of the
steroid hormones testosterone and estradiol as well
as the non-steroidal compounds hydroquinone and
b-hydroxybutyryl CoA. Pleurotus osteratus 17bHSD (17b - hydroxysteroid dehydrogenases) was
found to be pluripotent enzyme capable of
testosterone and hydroquinone oxidation. It thus
joins pluripotent HSDs whose role in detoxification
of xenobiotic carbonyl compounds in addition to
their role in the metabolism of endogenous steroids
and quinones is only suspected (Iwata, 1989). In
this study we showed protein content increased in

The effect of steroid hormone on Pleurotus florida

the some hormone treatments in P.florida and

fruiting initiation promote in these treatments.

Fig. 1. Effects of esteradiol and progesterone treatmenmts on the mycelial growth in Pleurotus florida. a
Control. b 1.5 M progesterone. c 3.02 M progesterone. d 6.05 M progesterone. e 1.5 M estradiol. f 3.02
M estradiol. g 6.05 M estradiol.

Fig. 2. Comparison of colony's diameter in the different hormonal treatments in the 6 th and 7th days after
inoculation.

Fig. 3. Fruit body formation in the hormonal treatments. a control. b

1.5 M estradiol. c 6.05 M estradiol. d 3.02 M progesterone.

The effect of steroid hormone on Pleurotus florida

Fig. 4. Protein content in the different hormonal treatments in Pleurotus florida (est=17-estradiol;
pro=progesterone).

Fig. 5. Protein profile by SDS-PAGE in the estradiol and progesterone treatments in Pleurotus florida
(e=17-estradiol; p=progesterone), arrows show a 28KD band.

11

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Wolter, M., Zadrazil, R., Martens and Bahadir,
M.(1997) Degradates of eight highly condensed
polycyclic aromatic hydrocarbon by Pleurotus
florida insolid wheat substrates. Appl. Microbial.
Biotec. 48: 398-404.

The Study of Physiological Responses of Musa

acuminata var. Mas to
Interaction of Salinity and
Cadmium

Mozhgan Farzami Sepehr1, 2*, Jennifer Ann Harikrishna2, Norzulaani Khalid2,

1-Department of Biology , Faculty of Agriculture, Islamic Azad University Saveh Branch,
Saveh, Iran
2-Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science
University of Malaya ,50603 Kuala Lumpur ,Malaysia

ABSTRACT
Soil salinity affects plant growth and development due to harmful ion effects and water stress caused by
reduced osmotic potential in the soil solution. Furthermore, Cd is a pollutant that has been emitted into the
environment for decades. Major anthropogenic sources are Cd-containing phosphate fertilizers, sewage
sludge and industrial emissions. Plants undergo one or more stress during their life cycle. The effects of
0,25,50 M Cd2+ (Cd(NO3)2.4H2O) and 0,50,75,100,125,150 mM NaCl on growth , the content of some ions
and proline contents in Banana (Musa acuminata var. Mas) were investigated in present study. With
increasing concentrations of Cd2+ or NaCl alone in culture media, growth parameters, Chlorophylls and
proline contents decreased. Combination treatment with salinity and cadmium decreased the negative effects
observed following the two stresses alone. Plants exhibiting growth retardation, none cadmium
accumulation in response to one mild stress factor (75,100,125 mM NaCl).the exposure of plants to cadmium
caused a partial reversal of effect of salinity. Root and shoot growth, ion accumulation, sensitivity index and
other physiological responses were improved at moderate concentrations of two stress factors imposed
jointly.
Key words: Musa acuminata var. Mas ,cadmium, salinity, growth parameters, ion accumulation, sensitivity
index,
Farzami Sepehr M.* and Harikrishna , J. and Khalid ,N (2010) The study of physiological responses of
Musa acuminata var. Mas to interaction of salinity and cadmium . Iranian J of Plant Physiology, 1(1):13- 22.

INTRODUCTION
Salinity is among the major stress that adversely
affect plant growth and crop productivity. This
constraint remain the primary causes of crop losses
worldwide , reducing average yields by more than
50% ( Boyer , 1982, Wang et al , 2003). Salt
induces osmotic stress by limiting absorption of
water from soil, and ionic stress resulting from high
concentrations of potentially toxic salt ions within
plant cells (Munns ,2002). Plants have evolved a
variety of protective mechanisms to allow them to
* Corresponding author: farzamisepehr@iau-saveh.ac.ir

Tel : +98-912-3803218
Received: July, 2010

Accepted: September, 2010

cope with these unfavorable environmental
conditions for survival and growth, including the
accumulation of ions and osmolytes such as praline,
the accumulation of these compounds prevents
water loss and ion toxicity. Farzami Sepehr and
Ghorbanli (2006a) found that, with increasing of
salinity till 170 mM NaCl at culture medium, the
proline and protein contents; Na and Cl amounts
significantly increased in Atriplex canescens L. Salt
stress tolerance in plants is a complex phenomenon
that may involve developmental changes as well as
physiological and biochemical processes(Hare and
Cress , 1997). The salt tolerance is a result of

inorganic ion accumulation , mainly Na and Cl

which are compartmentalized in the vacuole , while
organic solute accumulate in cytoplasm balancing
waterpotential throughseveral cellular compartments
(GreenwayandMunns,1980; Marschner , 1990 ;
Robinson et al, 1997; Serraj and Sinclair ,2002).In
addition to their role in cell water relations , organic
solutesaccumulationandalsocontribute to the
maintains of ionic homeostasis and stabilization of
some macromolecules and organelles such as
proteins,proteincomplexes and membranes
(Bohnert and Shen 1999; Bray et al 2000).
Cadmium (Cd) is a divalent heavy metal cation
and is one of the most toxic heavy metals with no
described physiological function. It enters the
environment through industrial processes and to a
lesser exert from natural weathering( di Toppi and
Gabbrieli , 1999). Although not essential for plant
growth , this metal is readily taken up by roots and
translocated into aerial organs where it can
accumulate to high levels. It has been shown that
Cd interacts with the plants water balance( Barcelo
& Poschenrieder , 1990 , Costa and Morel , 1994),
inhibits stomatal opening (Barcelo &
Poschenrieder , 1990), lower chlorophyll
content(Larsson et al, 1998), reduces growth ( Chen
and Kao , 1995), damages the photosynthetic
apparatus( Krupa , 1988, Sidlecka and Baszynsky ,
1993), and produces oxidative stress( Chein et al ,
2001; Hendry et al, 1992; Somashekaraiah et al ,
1992) . In spite of the considerable literature on the
subject, the mechanisms of Cd toxicity are not
known with any certainty.
Plants undergo one or more stresses during their
life cycle. Although most research has focused on
the responses of plants to a single stress factor,
plants in nature often meet multiple stresses, the
interaction of which may be far from
additive( Chapin et al , 1987).However , in some
cases preconditioning to one stress factor may
increase the tolerance of plants to another stress
factor imposed simultaneously or later( Farzami
Sepehr , Ghorbanli , 2006b).There are a few
literature about effect of salinity and cadmium alone
and with together on Banana plants. The aim of the
present study is to investigate the combined effects
of salinity and cadmium on growth, ionic,
chlorophylls contents and proline accumulation of
Musa acuminata var. Mas plants.

MATERIAS AND METHODS

Plant material and culture

Banana (Musa acuminata var. Mas) plantlets that

had produced from suckers of banana plants with 6
months olds collected from MARDI (Malaysian
Agricultural Research & Development Institute and
transferred to Plant Biology Incubator Unit of
University of Malaya. The plantlets were
transferred to new jars on Murashige and Skoog
medium supplemented with 30g/L sucrose , 50 g/L
Myo-inositol, 8g/L agars , NaCl [ 0. 50 , 75 , 100 ,
125 , 150 mM], Cd+2 [0, 25,50 M] immediately.
The initial pH of the medium was adjusted to 5.8
before autoclaving. The cultures were incubated at
253C with 16h light/ 8h dark with a light
intensity of 400 moles photo m-2s-1. After 30 days
of treatments plantlets from 6 containers were
removed. Plants were harvested at the beginning at
treatments (initial harvest) and after 4 weeks of
treatment (final harvest).
Growth and water content
In addition of Fresh weight (FW), Dry weight
(DW) was determined after desiccation at 70C for
48h. Leaf water content [WC, ml/g DW] was
estimated using the equation:
Equation 1: WC=(FW-DW)/DW (Chars et al , 2008)

Sodium,calcium,cadmium and praline

determination
Na+, Ca+2 and Cd+2 were assayed by atomic
absorption spectrophotometry after HCl: HNO3
(1:9) extraction of the finely ground dry matter
(Moragan, 1993). Proline was extracted and
estimated to Bates et al. (1973).
Chl a and Chlb determination
Chlorophylls were extracted with 80% acetone under
a dimgreen lightanddetermined spectrophotometrically
(Arnon , 1949).
Parameters for growth and nutrition analysis
The accumulation of dry matter upon treatment
depends on initial plant size , duration and growth
rate. Relative growth rate (RGR) eliminates
differences in biomass production related to
treatment duration and/or initial plant size (at the
beginning of treatment) (Chars et al, 2008).For such
reasons RGR gives a relative basis for comparison
of the effect of salt on plant growth among species
and genotype (Hunt , 1982). Plant RGR was
estimated as equation 2.
Equation 2: RGR= Mt
Where refers to the difference between values
at the final and initial harvest, t is the time (days)
and M is the whole plant DW (g). Ln is the
logarithmic mean of M calculated over the t period
(Hunt, 1990) according to the equation 3.

Response of Musa acuminata var. Mas to interaction of salinity and cadmium

Equation 3: M=M/Ln (M)

The sensitivity index (SI), i.e., the differences
between dry matter production of treated and
control plants, expressed in percent of the latter was
calculated according to equation 4:
Equation 4: SI treatment = [100( W treatment W control )]/ W control

This parameter was more negative when the plant

was sensitive to NaCl or Cd (Saadallah et al ,
2001).
Unite leaf rate (ULR) is synonymous with net
assimilation rate (NAR) (Hunt , 1999).ULR applies
both to plants growing as spaced individuals and to
plants growing in closed stands and was calculated
according to the equation 4:
Equation 4: ULR=(W2-W1)/(t2-t1) (Ln LA2- Ln LA1)/(LA2-LA1)

Statistical analysis
Analysis of variance and mean comparison
procedures was used to detect differences between
treatments. Mean separation procedures were

15

carried out using the ANOVA test with SPSS

(ver.14) significant difference at (P< 0.05).

RSULTS
Table 1 shows that salinity and cadmium alone
both affect banana plants growth, but the
combination of Cd and salinity positively growth
and reduced the individual toxic effects of the
stresses at some concentrations. With increasing
salinity, leaf area, shoot and root dry and fresh
matter of Banana plants decreased. Increasing of
cadmium to culture media had a negative effect on
various indices. At moderate salinity (75,100,125
mM NaCl) and Cd concentrations (25, 50 M Cd),
all growth parameters improved (at some cases are
significantly) compared with other treatments
groups. Thus, at moderate salinity, the resistance to
Cd increased and metabolic indices improved for
example, Unite Leaf Rate, Relative Growth Rate
were higher than control and other treatment groups
( Table 2). The Chl in Banana plants (Table 2)
decreased at various levels of salinity and following
cadmium treatment but an interaction between the
two resulted in an increase significantly in Chl.

Table 1: Root and shoot fresh weights, dry weights and leaf water content of Banana plants under different
treatments of salinity and cadmium (Mean Standard Error).
Cadmium
Cd2+(M)
0

25

Salinity

RFW

RDW

SFW

SDW

WC

(mM)

(g/Plant)

(g/Plant)

(g/Plant)

(g/Plant)

(ml/g)DW

0.6130.87g

0.00650.003f

1.0390.03g

0.1140.0005h

10.322.01bcdeg

50

0.3480.004cde

0.00050.0003abc

0.40.04ef

0.0380.006g

9.280.79abcdefg

75

0.2850.009bcd

0.00150.0008abc

0.3640.016cdef

0.0320.018fg

100

0.2240.01ab

0.0030.0017a

0.2380.03bcdef

0.02520.0017cdef

125

0.3680.019def

0.0050.0028de

0.2480.006ab

7.501.45abc

150

0.020.0003bcde

0.4690.035f

0.0210.012f

0.3590.012cdef

0.03160.0012fg

7.331.00abc

0.3090.047bcd

0.0110.006cd

0.2320.009ab

0.02620.0027def

6.520.59a

50

0.6130.033def

0.00610.0035abc

0.2010.09a

0.0370.0015g

7.142.54ab

abc

ab

75

0.2510.016

100

0.230.025ab

125

0.2270.008

ab

0.3170.055

bcd

150

0.00360.002

bcde

8.690.59abcdef

8.050.95abcd

0.3240.011bcdef

0.02060.0016

0.0020.0012a

0.430.06f

0.0310.0018fg

10.991.1cdefg

0.0080.0047abc

0.2330.003ab

0.01260.0003a

8.681.24abcdef

0.2240.015ab

0.01260.0006a

12.375.96fg

0.00940.0054

ab

11.670.73defg

50

0.1620.016a

0.0080.0046bc

0.2090.0085abcde

0.01930.0006abcd

6.981.55ab

50

0.3590.034cde

0.00490.0028ab

0.2680.011abcd

0.01660.0006ab

12.510.25g

75

0.2780.015bcd

0.0030.001abc

0.2560.025abc

0.0190.0015abc

100

0.3360.033bcd

0.00230.001abc

0.3660.029def

0.0220.001bcde

0.00830.004ef

0.3710.014def

0.0270.0023ef

0.00320.0018ab

0.2900.030abcd

0.0240.0023cde

125

0.4510.035

150

0.2260.004ab

ef

9.651.25abcdefg

12.032.5efg
8.430.87abcde
8.440.81abcde

Values followed by at least one same letter do not differ significantly at P <0.05 (n=3SE).
Table 2: Effects of combined Cd and NaCl treatments on some metabolic indices in Banana plants
Ion content
(ppm/gDW)

NaCl
(mM)

Cadmium content(M)

Chl a (mg/g FW)

0
50
75
100
125
150

0
0.610.02e
0.430.01cd
0.130.004a
0.480.2cde
0.3302bc
0.54.006de

Chl b(mg/g FW)

0
50
75
100
125
150

0.160.02a
0.160.003a
0.210.07a
0.360.05a
0.120.004a
0.320.04a

0.180.02a
0.180006a
0.300.02a
0.100.009a
0.110.004a
0.220.03a

0.130.006a
0.130.008a
0.760.35b
0.160.04c
0.870.01b
0.900.01b

Proline content
(mg/g leaf FW)

0
50
75
100
125
150
0
50
75
100
125
150

66.930.75a
87.460.40bc
130.771.2h
152.111.96i
175.61.87j
194.751.79k
0.0330.001i
-0.01150.0001h
-0.02120.0007g
-0.06530.001a
-0.04270.001e
-0.01840.0002g

88.203.18bc
97.942.26e
118.021.88g
189.703.66k
193.002.84k
203.243.23l
-0.03370.0008f
-0.03930.002e
-0.04970.0008d
-0.03170.002f
-0.06630.0008a
-0.06970.0008a

94.882.67cde
90.502.00cd
81.992.10b
107.310.63f
89.584.13cd
96.111.91de
-0.05700.001b
-0.06800.001a
-0.05430.002bc
-0.05230.001cd
-0.03070.001f
-0.05700.001b

RGR (g/gday)

25
0.350.03bcd
0.370.01bcd
0.660.01e
0.490.02cde
0.370.01bcd
0.350.003bc

50
0.370.01bcd
0.240.007ab
1.680.03g
4.080.08h
1.440.04f
1.410.01f

Response of Musa acuminata var. Mas to interaction of salinity and cadmium

ULR(g /m2day)

SI

Leaf
Area(cm2/plant)

0
50
75
100
125
150
0
50
75
100
125
150
0
50
75
100
125
150

0.040.002j
-0.0090.0005i
-0.020.001gh
-0.040.001cde
-0.040.001e
-0.020.0005h
------59.923.65cd
-63.380.54bc
-73.041.55a
-59.381.58cd
-45.357.16e
7.910.5abcd
9.520.41cdef
9.710.22def
8.260.88abcde
8.360.29abcdef
9.280.25bcdef

-0.030.001f
-0.030.0008f
-0.040.0005e
-0.020.001fg
-0.060.005a
-0.040.0005e
-60.473.65cd
-60.471.27cd
-75.042.02a
-69.941.66ab
-73.582.64a
-76.323.11a
7.350.49abcd
11.160.08fg
12.601.65g
13.151.36g
7.490.67abcd
6.660.08abc

17

-0.040.001de
-0.060.001a
-0.050.002bc
-0.050.001bcd
-0.030.001fg
-0.050.0008b
-67.942.21abc
-74.681.73a
-72.491.01a
-70.301.31ab
-52.093.17de
-70.120.36ab
10.920.98efg
7.441.24abcd
6.080.79a
7.970.52abcd
6.370.14ab
7.341.84abcd

Values are the mean SE. Values with different superscript letters are significantly different at the 5% level
(two ways Anova).
Concentration of Ca and K (Table 3) were lowered
following treatment with salinity or cadmium alone.
However cadmium treatment caused a significantly
improvement of Ca accumulation at intermediate
salinity levels. With increasing salinity in the
culture media, Na absorption rose significantly in
the shoots and roots of Banana plants, but Na was
accumulated more in shoots than in roots. Cadmium

uptake by Banana plants also decreased

significantly with increasing salinity, interestingly at
moderate concentrations of salinity there are no
absorption of cadmium from media. The roots of
Banana plants absorb and then accumulate the
cadmium; transportation of cadmium to shoot part
is low (Table3).

Table 3: Effects of combined Cd and NaCl treatments at ion content in Banana plants
Ion content
(ppm/gDW)

NaCl
(mM)

Cadmium content(M)
0

Cd content in roots

0
50
75
100
125
150

0.00a
0.00a
0.00a
0.00a
0.00a
0.00a

25
17.1810.219i
44.1670.167m
15.605 0.049g
13.25 0.055f
5.716 0.045b
6.813 0.008c

50
21.227 .107j
22.667 0.083k
26.41 0.164l
16.8940.161h
10.486 0.075d
10.9090.041e

Cdcontentin shoots

0
50
75
100
125
150

0.00a
0.00a
0.00a
0.00a
0.00a
0.00a

2.6350.015d
3.778.0277e
0.00a
0.00a
0.00a
1.4900.019b

28.3330.119l
10.1510.03i
7.2090.024h
5.2910.024g
2.0090.018c
4.9520.047f

Ca content in roots

0
50
75
100
125
150
0
50
75
100
125
150

314.40 4.17k
344.491.15l
138.083.03a
360.009.51m
281.424.52j
141.331.25ab
696.115.61i
330.981.67b
424.5314.18f
336.883.05bc
602.6614.81g
370.02.52de

190.760.57f
485.662.02n
219.870.89g
197.971.65f
190.120.53f
145.200.9abc
350.470.82bcd
650.8315.46h
373.111.45e
806.255.72j
897.389.19k
331.171.35b

148.461.44bc
152.360.4cd
232.720.9h
246.360.69i
159.331.07d
171.033.1e
358.451.25cde
377.422.1e
344.560.49bc
347.634.52bc
267.652.77a
1315.711.42l

0
50
75
100
125
150
0
50
75
100
125
150
0
50
75
100
125
150

166.660.83b
180.471.03c
340.431.09f
352.000.65g
564.602.55m
590.661.01n
172.640.58b
247.220.32d
282.001.17f
320.660.13h
600.882.11m
865.664.84p
180.890.61k
113.030.54i
109.850.32h
80.740.24e
76.110.55d
52.480.11b

93.610.68a
282.430.48d
359.621.49h
402.003.21j
428.880.77k
369.401.37i
229.360.15c
457.550.32k
545.003.36l
738.231.17n
773.090.13o
982.500.58q
181.120.64k
115.570.22ij
96.180.44fg
76.30.13d
71.570.23c
45.360.08

96.460.69a
180.352.28c
332.190.41e
353.791.89g
435.300.75l
436.661.18l
180.234.84b
266.667.42e
304.810.15g
357.572.11i
391.253.36j
609.953.81a
213.141.5h
95.660.18f
114.590.43ij
116.160.46j
98.670.18g
52.203.1b

0
50
75
100
125
150

49.760.39m
36.220.43h
32.530.19f
28.600.21d
25.090.04c
15.110.06a

45.110.06l
42.200.02k
39.110.07i
31.450.19e
32.310.12f
22.450.27b

55.520.25n
58.510.12o
45.380.23l
40.340.15j
34.360.14g
28.300.12d

Ca content in shoots

Na content in roots

Na content in shoots

k content in shoots

k content in roots

Values are the mean SE. Values with different superscript letters are significantly different at the 5% level
(two ways Anova).

Response of Musa acuminata var. Mas to interaction of salinity and cadmium

19

DISCUSION
Salinity inhibits plant growth for two reasons: First,
water deficit and Second due to salt-specific or ion
excess effects (Munns et al , 2006).Different plant
species have developed different mechanisms to
cope with these effects (Munns 2002). In this
research , reduction in plant fresh weight (root and
shoot), plant dry weight(root and shoot) , water
content , Relative growth rate and Unit leaf area
with increasing of salinity were observed.
Decreasing of growth at high amount of salinity in
media is corroborating the results obtained by Chars
et al (2008) in Arabidopsis and Thellungiella plants.
Salt tolerance has usually been assessed as the
percentage biomass production in saline versus
control conditions over a prolonged period of time
(Munns et al, 2000). Plants submitted to high
salinity decreased both root and shoot dry masses.
Once results demonstrate that root of young banana
plants is more sensitive to salinity than shoot
systems. According to Munns (1993), this sensivity
could be explained due to an imbalance among
cations as a result of the complex interaction in the
xylem transport system. By comparison of Na +
amount at shoot of young banana plants this
phenomenon could be associated to both a faster
osmotic adjustment and a slower turgor loss in
shoots ( Shalhevet et al 1995).Leaf area is the most
sensitive growth parameter in response to increasing
of salinity level in nutrient solution. In our results,
significantly decreasing of leaf area was not
observed by increasing of salt amount at media.
Leaf area is a function of leaf size; these results
suggest that the turgor adjustment was happened at
shoot therefore leaf size did not decrease. The
results showed depressive action of NaCl on water
content (WC), this behavior was generally
concomitant with a reduction in growth rate,
according to several studies salt in nutrient solutions
may inhibit plant growth by reducing plants ability
to take up water , leading to slower growth(osmotic
effect)and /or injuring cells in the transpiring leaves
(salt- specific or ion-excess effect)(Munns ,
2005).This was also true in the present study, as
shown by the negative relationship between Na+
tissue concentration and the plant hydration in
treated Banana plants. This negative relation
between water and Na+ contents also suggests that
banana may be deprived of efficient systems for Na +
vacuolar compartmentation and has an apoplastic
accumulation of Na+ in leaves. Data obtained by
Vera-Esrtella et al(2005) and Chars et al (2008)

support our hypothesis. This is a common response

observed in several glycophyes (Munns, 2002).
With increasing of salinity in Banana treated plants
disturbance of K+ nutrition appeared. This could
result from two factors: a) inhibition of root growth
and b) decreased intrinsic capacity of root for ion
uptake (uptake efficiency). The decrease of
K+ uptake efficiency could be direct competition
between K+ and Na+ for root transporters. Because
of the similar physicochemical properties of both
ion, Na+ at high concentrations has a strong
inhibitory effect on K+ uptake by the root ( Fu and
Luan , 1998, Kim et al 1998).Although several roles
have been attributed to accumulation of proline
upon stress, its role in plant stress adaptation is still
a subject to debate. Interestingly, the overexpression in tobacco of a modified P5C5 gene
involved in proline biosynthesis from Vigna
aconitifolia conferred higher generation rates
increased biomass and production to fewer free
radicals upon salt stress(Hong et al , 2000). In our
results, proline accumulation increased in Banana
plants under salt stress. In the same context, a large
increase in proline was found by Andrade et
al(1995) in leaves of four bean cultivars subjected
to drought stress, with the drought susceptible
cultivars accumulating more of this osmolyte. A
negative relation between salt tolerance and proline
accumulation has also been reported ( Petrusa ,
Winicov, 1997, Nanjo et al , 2003).In this
physiological backgrounds, proline accumulation
appears as a consequence of a disturbance of cell
homeostasis and/or of increase in the use of
photosynthesis products for proline biosynthesis at
the expense of plant growth.
Cadmium is a certainly and effective inhibitor of
plant metabolism, particularly of photosynthetic
processes and chloroplast development in higher
plants ( Rascio et al ,1993).This was confirmed in
the present experiments : Cd 2+ primarily affected
the contents of photosynthetic pigments unless
otherwise indicated Cd toxicity in leaves caused by
excess Cd was indicated a decrease in chlorophyll
contents. Effects of Cd on chlorophyll and
chloroplast development act synergistically and
inhibit photosynthesis (Rascio et al 1993, Prasad ,
1995). An excess of metal ions in plants can induce
a series of effects which present some common
characteristics: alternation of water status and
inhibition of root growth ( Hagemeyer and Breckle,
1996 , Prasad and Hagemeyer, 1999).The results

show to decreasing of water content and growth of

Banana plants with increasing of cadmium at
nutrient media . Toxic levels of Cd (Barcelo et al
1988a, Paivoke , 1983) have been found to decrease
the vessel diameter in diverse plant species. In bean
plants stems 44.5M Cd decreased both the vessel
radius and the numbers of vessels, resulting in
decreased total vessel area and a more than 50%
decrease of the sap flow rate (Barcelo et al 1988b).
The decrease of both number and size of vessels in
Cd treated plants may be caused by the inhibition of
cell elongation (Barcelo et al 1988b). If important at
all, the metal induced decrease of the xylem
capillary radius may only play a role after relatively
long metal exposure while alternations of root and
leaf water relations can be observed after a few
hours(PrasadandHaegemeyer,1999). With increasing
of Cd at media , proline contents at Banana leaves
increased. Mehta and Gaur (1999) showed that
proline accumulation and proline pre-treatment
prevented metal-induced lipid peroxidation and
potassium ion efflux. With respect to increasing
proline accumulation and K+ content, proline
protective function seems to be probable in Cdtreated banana plants. Higher levels of Cadmium in
nutrient solution reduce the Ca contents as a result
of the competition between Cd and other bivalent
cations (Karez et al 1990 , Farzami Sepehr and
Ghorbanli ,2006). CdClx complexes are formed in
the water and these complexes are hardly taken up
by plants (Prasad and Haegemeyer, 1999), because
of this, cadmium uptake with increasing salinity
decreased and the negative effects of salinity on the
uptake of Ca, K, Na, growth processes and Chl
contents were alleviated and plant responses were
improved.
Some reports have indicated that imposing drought
and salinity stress on plants may render them more
tolerant to later damage by environmental pollutants
(King and Nelson ,1987 , McBirde, 1987).The
results of the present study indicate that plant whose
growth has been retarded by a mild first
stress(25,50,75,100 mM NaCl) may become more
tolerant to a second stress(Cadmium).An interaction
between cadmium and salt stress increased
RGR,ULR, Leaf Area, RFW,RDW,SFW,SDW( at
some cases)and Chl contents.These results are
agree with Prasad(1995) findings. The comparison
of sensivity index(SI) between different treatments
of salinity , cadmium(alone) and interaction of two
treatment showed that the sensivity of Banana
plants to salinity is lower that Cd and at interaction

of both treatments SI a little decreased [at media

that supplemented by 125mM NaCl and Cd].
In conclusion, the presence of cadmium in nutrient
solution in combination with NaCl affected the
responses of plants. At moderate levels of cadmium
&NaCl , all of physiological responses of Banana
plants that were studied improved compared which
other treatments , namely salinity & Cadmium
alone. The cadmium influx into plants decreased in
the presence of salinity, thus reducing the harmful
effects of salinity and cadmium observed.

ACKNOWLEDGMENTS
This work was supported by PBIU( Plant
Biotechnology Incubator Unit) and CEBAR(Centre
for Research in Biotechnology for Agriculture)
programs in University of Malaya, Kuala Lumpur ,
Malaysia. Special thanks from Prof. Dr. Yasmin
Othman for her kindly cooperation.

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Electromagnetic Fields Effect on Photosynthetic

Pigments, Parietin and Proline of two Lichen Species
Mahlagha Ghorbanli*, Talayeh Amirkian and Mohamad Ali Rezaei
Department of Biology, Faculty of Science, Islamic Azad University Gorgan Branch, Gorgan, Iran.

ABSTRACT
This study is aimed to evaluate the effects of electromagnetic fields (B=15, 23 mT(AC)), at 50 Hz frequency
on Xanthoria parietina and Lepraria lobificans. Chlorophyll a, chlorophyll b, parietin and proline in both
species treated with electromagnetic fields 15, 23mT decreased in compare to control. Only chlorophyll a in
L. lobificans and xanthophyll in X. parietina in treated with electromagnetic fields 15mT increased in
comparison with control. Proline compound in X. parietina increased in treated with electromagnetic fields
23mT in compare to control. The results indicated that different periods of electromagnetic fields cause
physiological response in lichens.
Key words: electromagnetic fields; photosynthetic pigrnenis; proline; parietin.
Ghorbanli M.* ,Amirkian , T and Rezaei, M.A. (2010) Electromagnetic Fields Effect on Photosynthetic
Pigments, Parietin and Proline of Lichen Species . Iranian J of Plant Physiology, 1(1): 23-29.

INTRODUCTION
Lichens are slow growing symbiotic organisms. The
symbiosis is between fungus (mycobionts) and a
photosynthetic partner (photobionts). The later
could be a green algae or cyanobacteria. Lichens
dont possess roots or waxy cuticle. They are
mainly dependant on the atmospheric input of water
and mineral nutrients. Consequently, the entire
thallus area of lichens is susceptible to penetration
and accumulation of airborne elements, some
essential for proper functioning of the lichen but
others are toxic (Weissman et al., 2005). Lichens
are typical pioneers of the environment because of
their ability to survive in extreme and inhospitable
conditions, such as drought, low/high temperature,
low/high irradiance, etc (Bartak et al., 2008). More
than 800 different lichen compounds have been
isolated and identified, being deposited as numerous
tiny crystals outside living fungal hyphae (Solhaug
et al., 2009). Lichens may contain substantial
amounts of secondary compounds, up to 30% of the
dry weight (Huneck, 1973). Parietin is an orange
*Corresponding author: mghorbanli@gorganiau.ir
Tel : +98-21-44364066
Received: July, 2010
Accepted: September, 2010

coloured anthraquinone pigment located as tiny

extracellular crystals in the top layer of the upper
cortex of the members of the lichenised fungal order
Teloschistales to which the foliose lichen Xanthoria
parietina belongs (Gauslaa & McEvoy, 2005). The
fungal synthesis of parietin is induced by UV-B
(Solhaug et al., 2003) and stimulated by
photosynthates which provided by the symbiotic
green algal photobiont Trebouxia, resulting in a
close, positive correlation between habitat- specific
solar exposure and parietin content of X. parietina
(Gauslaa & Ustvedt, 2003). Numerous experiments
proved that the static or extremely low frequency
magnetic fields with small flux density had an effect
on various living organisms (Pal, 2005). A
considerable part of the investigation dealt with the
effect of electromagnetic fields on macromolecules
or cells (Pal, 2005). It is well known that magnetic
fields produce biochemical, physical and
physiological changes in cell structures
(Pietruszewski et al., 2007). Also it is clear that
static and variable magnetic field may have a
positive, but mostly temporary and impermanent
effect on the percentage of germination, growth rate
and germination rate (Pietruszewski et al., 2007).

Also it was shown that bean sprouts growing in an

electric field have a better growth rate height of
stems and length of roots in comparison to nonexposed seeds (Kiatgamjorn et al., 2002). The effect
of 200mT flux density static and 29mT flux density
pulsating magnetic field on the different species of
fungi, according to their examination, morphological
changes were observable on the conidia of
Aspergillus puniceus and Alternaria alternata, the
pigmentation of the colony of Aspergillus niger
changed, the culture and remained white (Sadauskas
et al., 1987). Electromagnetic field of 0.05 to 0.30T
on the seeds of wheat, barely and oat showed that
magnetic fields had a positive effect on the
germination rate as well as root and shoot lengths
(Bhatnagar& Deb, 1977). The main objective of this
study is to evaluate the influence of electromagnetic
fields on physiological response of X. parietina and
L. lobificans and to further investigate some aspects
of lichen physiology in more details.

frequency, 150v and 0.24A for 4 hours during a 2 or

3 days period. Lichens grow naturally in the earths
magnetic field which its intensity is 0.5G. The
device used in this experiment was able to produce
stronger magnetic fields than earths magnetic field.
This devise successfully investigated the tolerance
and flexibility of physiological reactions. During
this experiment the room temperature was kept
between 24 to 26 C. Electromagnetic field
intensity resulted from this equation:
NI
B 0 r
L
o = The permeability factor in vacuum which is
equal to 4 10 7
r = The nuclear permeability factor in 100v is
about 13128 and in 150v is about 12491.

MATERLALS AND METHODS

B = magnetic field according to the Tessla (T).

Sample collection

Pigment assays

Epiphytic lichen L. lobificans Nyl. was collected

from a forest experimental site in north of Iran
which is called Laffor with 52 49' E longitude
and 3612'N Latitude from rocks bryophytes with
four replications. Another epiphytic lichen X
.parietina (L.) Th. Fr. was collected from north
shore region of Iran (in southern part of Caspian
Sea) with 36 43'N latitude, 52 39'E longitude and
-21m elevation from Platanus orientalis with four
replications.

Total chlorophyll content was determined using

Arnons method (Arnon, 1949). The level of
absorbance was measured using
spectrophotometrically at 663nm for chlorophyll a
and 645nm for chlorophyll b wavelength against
blank samples. Chlorophyll content was estimated
base on mg. g 1 FW.
Carotenoid was estimated using Jenson (1987)
methods. Its absorbance was measured
spectrophotometrically at 445nm for xanthophyll
and 450nm for carotene wavelength. Carotenoid
content was calculated base on mg. g 1 FW.

Producing electromagnetic Fields

Because of the size of lichens, a plastic spool at
44cm was used. For achieving strong magnetic
fields, a device by the use of wire with 0.4mm
diameter and 1688m length was made, so 10550
helix wires have coiled regularly. Dimmer for
providing alternative electricity (AC) with low
voltage also was applied. This device has adjustable
resistance in different ranges that can reduce the
local electricity (P. N. 220v-50Hz). Two nuclear that
made up Ferromagnetism and its cross section is
about 8 cm2 and4/9 cm2, for limiting the electrical
current of the wire coil were fixed. Lichens were
located in the middle of the spool until they have
normal contact with magnetic field lines. The
direction of the magnetic fields was changed 50
times in a second. In the first stage, lichens were
stressed by electromagnetic fields 15mT at 50Hz
frequency, 100v and 0.15A, for 4 hours in a 2 or 3
days period. In the second stage, lichens were
stressed by electromagnetic fields 23mT, at 50Hz

N= The number of helix coil wire.

I= The current intensity that is passing through the
wire coil is 0.15, 0.24 according to Amper
L = Wire coil length according to Meter (m).

Proline assay
Proline was estimated by Bates et al., (1973)
methods. The absorbance was measured
spectrophotometrically at 750nm. Proline was
calculated base on Mol. g 1 FW.

Parietin assay
Parietin was determined by Solhaug &Gauslaa
(2001) methods. The absorbance was measured
spectrophotometrically at 434nm. Parietin was
calculated base on OD. min .1 g 1 DW .
Statistical analysis

Electromagnetic effect on Lichen species

Data were analyzed using SPSS software (version

15). One way analysis of variance (ANOVA) and
the statistical significance of the results were
analyzed by using Duncan test. The charts were
designed by using Excel software.
RESULTS
Total Chlorophyll assay
The results from chlorophyll assay indicated that
chlorophyll a in X. parietina was decreased during
electromagnetic fields 15, 23mT significantly at
0.05 levels in compare to control. The results also
indicated that the amount of chlorophyll a in L.
lobificans was increased during electromagnetic
field 15mT significantly at 0.05 levels in compare
to control (Fig. I). Chlorophyll b in X. parietina was
decreased significantly at 0.05 levels during
electromagnetic field 15, 23mT in compare to
control. Chlorophyll b in L. lobificans during
electromagnetic field 23mT decreased significantly
at 0.05 levels in compare to control and 15mT stress
samples (Fig. II).
Carotenoid Test

25

The amount of carotene in X. parietina during

electromagnetic field 15mT decreased
insignificantly at 0.05 levels in compare to control
and in 23mT stress condition increased
insignificantly at 0.05 levels in compare to control.
Also the amount of carotene in both 15, 23mT
electromagnetic fields increased insignificantly in
compare to control in L. lobificans (Fig. III).
Xanthophyll in X. parietina during electromagnetic
fields 15, 23mT increased significantly at 0.05
levels in compare to control. In L. lobificans the
amount of xanthophyll just during electromagnetic
field 23mT increased significantly in compare to
15mT stress sample (Fig. IV).
Analysis of Proline Level
Proline content in X. parietina during
electromagnetic field 23mT increased significantly
at 0.05 levels in compare to control. But this content
in L. lobificans decreased significantly in both 15,
23mT stress conditions in compare to control (Fig.
V).
Parietin Test
Parietin in X. parietina during electromagnetic
fields 15, 23mT decreased significantly in compare
to control (Fig. VI).

Fig. I. Effect of electromagnetic fields on the chlorophyll a

base on mg. g 1 .FW is significant at p <0.05 in both species
except the increase of 23mT stressed sample in L.lobificans
and control sample had highest axis in X.parietina.

Fig. II. Effect of electromagnetic fields on the chlorophyll b

base on mg. g 1 .FW is significant at p <0.05 in both species
except the increase of 15mT stressed sample in L. lobificans.

Fig. III. Effect of electromagnetic fields on the carotene base on

mg. g 1 .FW is insignificant at p <0.05 in different ranges in both species.

Fig. IV. Effect of electromagnetic fields on the xanthophylls

is significant at p <0.05 the axis was normalized at its respective
highest peak in 15mT stressed sample in X.parietina.

Electromagnetic effect on Lichen species

27

Fig. V. Effect of electromagnetic fields on the proline base on

Mol. g 1 .FW. is significant at p <0.05 and the lowest
amount was shown in stressed samples in L. lobificans.

Fig. VI. Effect of electromagnetic fields on the base on

OD. min .1 g .1 DW . parietin is significant at p <0.05.

DISCUSSION
The analysis of modulated chlorophyll a fluorescence
in lichen is one of the several methodologies used
for the assessment of the level of environmental
stresses such as temperature, osmotic stress, heavy
metal and air pollution (Unal et al, 2009). Also, it
was determined that in lichens growing in their
natural habitat the chlorophyll content was higher at
the most polluted locations as an adaptation to air
pollution (Shukla and Upreti, 2007). These changes
are probably caused by the influence of air
pollutants on the integrity of chlorophyll a, which is
more sensitive to oxidations than chlorophyll b
(Gries, 1996). Moreover the Chl-b/ Chl-a ratio in U.
amblyoclada thalli increased in all the samples
transplanted to polluted zone, indicating a decrease
of Chl-a concentration due to degradation
(Rodriguez et al, 2007). Our findings do not
completely support the previous work, because in
electromagnetic fields not only the amount of
chlorophyll a but also the amount of chlorophyll b
was decreased in both 15 and 23mT stress
conditions in compare to control. In other studies in
electromagnetic fields with 900MHz frequency a
slight increase in chlorophylls ratio of Zea mays L.
young plantlets value was obtained for short
electromagnetic field exposure times a diminished
value for chlorophyll ratio was revealed (Racuciu
and Miclaus, 2007). Recent studies revealed that
carotenoids might also protect plants from oxidative
by modulating physical properties of photosynthetic
membranes with an involvement of the

xanthophylls cycle in this process (Gruszecki and

Strzalka, 1991). Carotenoid has significant positive
correlation with Cu, Cr and Zn; Copper in high
concentration can decrease total carotenoid
concentration in Trebouxia cell (Backor et al, 2003).
This idea didnt support carotene measurement,
because it was insignificant in both species. But this
finding support xanthophyll measurement because it
was increased in 23mT stress sample in L.
lobificans. In lichens containing green algae as the
photobiont, the photoprotective xanthophyll cycle is
of great importance (Herber et al, 2006). Proline
possibly acts as an antioxidant, and prevents lipid
peroxidation by reducing the free radicals damage
(Tripathi and Gaur, 2004).
Proline content increased in both in leaves and roots
after exposure of pea plants to high concentrations
of Ni (Gajewska and Sklodowska, 2005). Increase
in proline concentration has also been reported after
treatment of plants with Cu (Ali et al, 1998). This
idea supports our investigation, because proline
concentration in X. parietina increased in
electromagnetic fields at 23mT. There is now
substantial evidence showing that cortical lichen
compounds protect lichen photobionts against
excessive photosynthetically active light (McEvoy
et al, 2007). The photoperiod and solar power have
also increased sufficiently to cause a significant rise
in temperature, increasing temperature and
irradiance are likely to enhance lichen
photosynthesis and thus provide an increasing

amount of photosynthates from the photobionts that

stimulates fungal parietin synthesis (Gauslaa and
McEvoy, 2005). Our findings do not completely
support the previous work, in both electromagnetic
at 15 and 23 mT parietin decreased in compare to
control. Our findings demonstrated that
electromagnetic fields had significant effects on
lichen compounds, also indicated that lichen
physiological response towards electromagnetic
stress and eliminate some unclear points about
lichen physiology.

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29

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30

Study of Eucalyptus Allelopathy Effect on

Morphophysiological Parameters of Brassica napus L.
Maryam Niakan* and Kolsum Saberi
Department of biology. Islamic Azad University.Gorgan branch. Iran

ABSTRACT
In this research effects of aqueous extracts (0, 5%, 15%, 30%) and decompose of Eucalyptus leaf (in ratio 0,
3%,6% with soil) on growth parameters, chlorophyll a, b, soluble sugars and phenolic compounds content of
canola (Brassica nupus L) in pot condition were evaluated. The results showed that length, fresh and dry
weight of canola were decreased when exposed to different concentrations of decompose while aqueous
extracts of Eucalyptus leaf increased them. In addition aqueous extracts and decompose of Eucalyptus leaf
increased chlorophyll a , b and rate of them in canola leaf. The findings also indicated canola leaf treatment
with aqueous extracts of Eucalyptus increased the soluble sugars in comparison to the control but
decompose of Eucalyptus leaf reduced these compounds. On the other hand, Phenolic compounds in canola
leaf in response to Eucalyptus aqueous extracts were decreased while decompose of Eucalyptus leaf did not
have considerable effect on them.
Keywords: Brasica napus, growth ,allelopathy Eucalyptus, photosynthesis.
Niakan, M.* and Saberi, K. (2010) Study of Eucalyptus Allelopathy Effect on Morphophysiological
Parameters of Brassica napus L. Iranian J of Plant Physiology, 1(1): 30 - 36 .

INTRODUCTION
The importance of allelopathy in nature and in
agroecosystem has attracted researcher's attention.
Allelopathy, the chemical mechanism of plant
interference, is characterized by a reduction in plant
emergence or growth. Modern research suggested
that allelopathic effects can be both positive and
negative, depending upon the dose and organism
affected (Bais et al 2003)
The multiple effects resulting from allelopathic
allelochemicals include decreases in plant growth,
absorption of water and mineral nutrients, ion
uptake, leaf water potential, shoot turgor pressure,
osmotic potential, dry matter production (Booker et
al,2002 ;Gerald et al ,1992 ; Yang et al ,2004).
These gross morphological effects may be
secondary manifestations of primary events, caused
by a variety of more specific effects acting at the
cellular or molecular level in the receiver plants
(Hierro and Callaway, 2003; Prati and Bossdorf ,
2004; Weir et al 2004).
*Corresponding author :neda.niakan@gmail.com
Received: May, 2010
Accepted: August, 2010

Eucalyptus is one of the most important tree species

for wood production in world. It is said that
Eucalyptusistoxic (Shiva and Bandyopadhyay,1985).
Researchers showed that Eucalyptus species
released volatile compounds such as benzoic,
cinnamic and phenolic acids, which inhibited
growth of crops and weeds growing near it (Narwal,
1990; Suresh and Rai, 1987). Schumann et al.
(1995) reported that water extracts of Eucalyptus.
grandis significantly reduced weed establishment.
The aim of present study was to determine the
inhibitory effect of decompose and aqueous extract
of Eucalyptus leaf on growth, amount of Chla, b
soluble sugars and phenolic compounds in canola.

MATERALS AND METHODS

Decompose of Eucalyptus leaf preparation
Eucalyptus leaves (Eucalyptus camaldulensis)
were harvested in Azad Shahr city ( 34 m above sea
level) in North of Iran. Then leaves were dried in
shade and were grinded. Dried samples were mixed
with soil (Si-Clay texture) in ratio 0 as a control,

3% and 6% and this mixture was placed in shade for

30 days.
Aqueous extract of Eucalyptus Leaf Preparation
Five g of a dried Eucalyptus leave (Eucalyptus
camaldulensis) were added to 150 ml distilled water
and were shaked for 12 h. The mixture was passed
through Wathman paper (NO 2) and micro pore
filter (0.2 micron) and the aqueous extract in
concentrations of 0, 5%, 15% and 30 %was
prepared.
Canola Planted
Canola planted in decompose of Eucalyptus leaf
Canola seeds (Brassica napus L.cv Hyola 401) were
imbibed in distilled water for 48 h and then planted
in pots where include mixture of soil (Si-Clay
texture) and Eucalyptus leave in ratio of 0 as
control, 3% and 6%. Pots were maintained in a
photoperiod and temperature 20 2 C and 10h light /
14 h dark and irrigated with 200 ml water per 72 h.
After 60 days plants were harvested in soil and used
for growth and biochemical assays.
Canola planted for spray aqueous extract of
Eucalyptus leaf
The other numbers of canola seeds (Brassica napus
L.cv Hyola 401) also were placed in water for 48 h
and planted in pots soils with Si-Clay exture. After
30 days, canola plants were sprayed with aqueous
extract of Eucalyptus leave in concentrations of 5%,
15% and 30% for 3 times in a week during 30 days.
After this time, plants were brought out from the
soil and used for growth parameters measurement
and biochemical assays.
Growth Parameters Measurement
After 60 days, root and shoot length of canola plants
that had been treated by decompose and aqueous
extract of Eucalyptus leaf were measured. Fresh and
dry weight of root, shoot and leaf area of canola
plants under treatments of decompose and aqueous
extract of Eucalyptus leaf also were determined. For
dry weight, root and shoot of Canola were placed in
oven at 90 C for 24h.
Chlorophyll assay
Amounts of chlorophyll a & b in leaf of canola
seedling in treated and control were evaluated by
Bruisma, (1963). At first cotyledons were weigthted
and homogenized in 5ml acetone. then the mixture

was centrifuged at 3000 rpm for 15 minute and

supernatants were separated and rate of their wave
length absorption in 645, 652 and 663 nm with
spectrophotometers was measured.
Soluble Sugars Assay
To determine the soluble sugars, root and leave of
canola were dried in oven at 110 C for 48 h. They
were weigthed and 10 ml ethanol (70%) was added
then the samples were placed in Petri dishes for 7
days at 4 C. Soluble sugars contents were
determined by measuring the absorbance at 485 nm
spectrophotometrically with Kochert method,
(1978). Glucose standard curve was obtained to
estimate the soluble sugars concentration (mg g-1
DW).
Phenolic Compounds Assay
Phenolic compounds were extracted from 1 g fresh
plant material as described by Matta et al(1969) . In
this method, samples were boiled in 10 ml of 80%
alcohol for 15 min and then centrifuged for 15 min
at 3000 rpm. To 5 ml of this solution, 5 ml of
diluted folin (1:3) and 10 ml of saturation Na2CO3
were added. Samples were remained for 10 min at
25C and then centrifuged for 15 min at 4000 rpm.
Statistical Analysis
The statistical significance of the difference
between parameters was evaluated by means of
Duncan-test on SPSS (Version 11.5) and for each
treated and control 4 replications was selected. The
results are given in the text as p, the probability
values, and p0.01 was adopted as criterion of
significance.

RESULTS
The results showed that by increasing the amount of
decompose Eucalyptus leaf in soil (3%, 6%) length,
fresh and dry weight of canola root and shoot at
p0.01 were decreased (Table 1). While application
of higher amounts of aqueous extract of Eucalyptus
leaf increased the length, fresh and dry weight in
canola shoot, it did not have any significant effects
on fresh and dry weight of canola root at p0.01
(Table 2). Also the analysis of data revealed that
decompose of Eucalyptus leaf had more inhibition
effect on fresh and dry weight of root and shoot of
canola than aqueous extract of Eucalyptus leaf

(Tables1,2 ). Fig (1, A-D) shows the amount of chl a,

chl b, (a + b) and chl (a/b) in leaf canola under
different concentrations of decompose and extract of
Eucalyptus leaf. Amount of chl a and b increased
with rising decompose rate of Eucalyptus leaf in
soil. The most amount of Chl a and b was seen in
%15 concentration of Eucalyptus leaf extract. Also
to increase concentration of extract Eucalyptus, chl
and b content rose significantly. The our results
showed that soluble sugars content in canola leaf
and root also decreased with increasing content of
decompose of Eucalyptus leaf while their amount
increased in canola leaf under treatment of
Eucalyptus leaf extract in comparison with the
control (Figure II- E,F). Fig (II-G,H) shows the
amount of phenolic compounds in canola root and leaf
at different treatments. With increasing content of the
extract of Eucalyptus, level of phenolic compounds in
canola leaf increased significantly while decompose
of Eucalyptus did not have any significant effect on
the leaf. The results this research showed that canola
response to decompose and extract of Eucalyptus
leaf was different. Growth parameters in canola in
presence extract of Eucalyptus leaf increased while
decompose of Eucalyptus leaf decreased canola
growth. Canola leaf chlorophyll content in both
treatment increased. Extract of Eucalyptus leaf
increased soluble sugars in canola while decompose
of Eucalyptus leaf reduced them. Canola phenolic
compounds also decreased only in Eucalyptus
aqueous extract .

DISCUSSION
The studies have shown that the roots of plants
which exposed to allelochemicals became brownish,
stunted and void of root hairs that these changes
were also observed in our experiments ( the data is
not shown). This might be due to the rapid
inhibititory effect on respiration of root tips, which
ultimately reduced elongation (Shahid et al 2006).
Cao (1992) reported that aqueous extract from bark,
leaf and volatiles from leaf of Eucalyptus citriodora
showed allelopathic effect on the growth of 9
species including weeds like Bidens pilosa
,Eragrostis cilianensiss, Setaria geniculata, and
crops like corn, rice, cucumber, bean and
Stylosanthes guianensi.According to some researches
reduced biomass of Avena fatua, Convolvulus
arvensis, Rumex dentatus, Phalaris minor and
Triticum aestivum when exposed to different plant
water extracts of Sorghum, Sunflower, Eucalyptus
and Acacia might be the result of reduced dry

matter accumulation (An et al,1996; Rizvi and

Rizvi ,1992) .-Pinene is one of the major
components of volatiles released by a wide range of
species including Eucalyptus sp., Pinus sp. and
Quercus sp (Geron et al ,2003). -Pinene inhibits
seed germination and primary root growth in maize
(Abrahim et al ,2000) and disrupts energy
metabolism by acting as an uncoupler of oxidative
phosphorylation and inhibiting the electron transport
chain (Abrahim et al,2003).Lovett et al( 1989)
reported that biological activities of receiver plants
to allelochemicals are known to be concentration
dependent with a response threshold. Responses are,
characteristically, stimulation at low concentrations
of allelochemicals, and inhibition as the
concentration increases. Identical results were
reported by Anjum and Bajwa (2005) and Nasim et
al (2005). In this study, it seems that inhibitor
compounds content as -Pinene in decompose
Eucalyptus leaf were high and could reduce growth
parameters in canola while these compounds
concentration in aqueous extract of Eucalyptus leaf
were low and could stimulate growth in canola.
Researchers showed that Eucalyptus species
released volatile compounds such as benzoic,
cinnamic and phenolic acids, which inhibited
growth of crops and weeds growing near them
(Narwal ,1990; Suresh and Rai,1987). The phenolic
acids caused chlorophyll reduction in soybean and
sorghum seedlings (Einhellig and Rasmussen,
1979). Rice (1984) showed that allelochemicals
such as phenolic acid inhibit biosynthesis
chlorophyll precursors .Decreasing chlorophyll by
allelochemicals result in inhibition of chlorophyll
biosynthesis or induction of their degradation
pathway. It is thought that allelochemicals such as
phenolic acid induces activity of chlorophylase and
Mg - chelatase (Yang et al 2004). Our results were
different from the above researches. Since plants
responsedependsontheallelochemicals concentration
(Lovett et al, 1989), in this research it seems the
amounts these compounds in treatments related to
decompose and extract of Eucalyptus leaf were low,
thus they stimulated biosynthesis chlorophyll or
reduced degradation pathway in canola leaves.
Among so many symptoms, a decrease in
photosynthesis efficiency is a common effect of
allelopathic phenolics.Sorgoleone,ap-benzoquinone,
in Sorghum bicolor root exudates was found to
inhibit the oxygen evolution of soybean leaf disk
and isolated pea chloroplast, which in turn caused
growth reduction and photosystem II electron
transfer reaction (Einhellig et al, 1993). -

Coumaric, ferulic, cinnamic ,vanillic acids, and

coumarin severely suppress the photosynthesis of
soybean and Lemna minor L. Also the chloroplast
structure show a decrease in area, number and width
of grana, number of thylakoids in response to
allelochemical (Einhellig,1986).The phenolic
compounds are a large chemical group that plays
antioxidant role. Antioxidant molecules inactivated the

active oxygen species such as superoxide anion

radical O02 , H2O2, singlet oxygen (Apel and
Hirt,2004).It seems that only Eucalyptus leaf extract
in application concentrations had positive effect on
phenolic compounds in canola leaf.

Table(1): Effects of different concentrations of decompose of Eucalyptus leaf (in ratio 0=control , %3, %6
with soil) on root and shoot length (cm), dry and fresh shoot and root (g) of canola.. Means SD
followed by unlike letter of the same column indicates that the values are significant different at 0.01
determined by ANOVA and Duncan multiple range test.

Root
Control
Decompose (3%)

Length
7.8a

Decompose (6%)

F.W
1.493a

5.567b

Shoot
D.W

Length

F.W

0.293a

3.333b

4.877ab

0.809b

4.917b

0.121b

0.271c

0.045c

D.W
0.879a

4.833a

5.938a

0.561b

3.483b

2.074b

0.193c

Table(2): Effects of different concentrations of aqueous extracts (0, %5,%15,%30) of Eucalyptus leaf on root
and shoot length(cm), dry and fresh shoot and root (g) of canola. Means SD followed by unlike letter
of the same column indicates that the values are significant different at 0.01 determined by ANOVA and
Duncan multiple range tests.

Root
Control
aqueous extracts (5%)
aqueous extracts (15%)
aqueous extracts (30%)

Length
9.875 ab

Shoot

F.W

D.W

Length

F.W

2.138a

0.339a

6.667c

4.379a

0.709ab

5.788c

3.648a

0.649b

9.238b

5.109a

0.773ab

8.838 b

1.695a

0.3a

9.143 b

1.376a

0.308a

10.975 a

1.609a

0.32a

11.825a

5.906a

D.W

1.049a

(A)

(B)

(C)

(D)

Fig ): Effects of different concentrations of decompose ( in ratio 0=control , %3, %6 with soil) and extract
of Eucalyptus leaf (0=control, %5,%15,%30)) on chlorophyll a,b (A, B) and chl a+b , a/b in canola leaf
(C,D,E,F). Bars indicate the standard deviation

(F)

(E)

(G)

(H)

Fig ): Effects of different concentrations of decompose (in ratio 0=control , %3, %6 with soil) and extract
of Eucalyptus leaf (0=control, %5,%15,%30) on amounts of soluble sugar(E,F) and phenolic compounds
(G,H ) in leaf and root of canola. Bars indicate the standard deviation

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37

Relationship Between Some Nutrient Uptake and

Early Abscission of Fruits in Ash (Fraxinus excelsiorL.
)

Farhang Moraghebi 1 Baba Khanjanishirazi 2 and Maryam Teimouri 3

1 : Biology Department. Science faculty. Islamic Azad University Shahr-e- Rey Branch.
2: Guilan Research center for agriculture and natural resources
3: Forest Research Division. Research Institute of Forests and Rangelands. Tehran. Iran.

ABSTRACT
Ash (Fraxinus excelsior) has a distribution from Astara in Guilan to Gildaghi in Golestan province in North
part of Iran. This species is used widely in reforestation programs because of its suitable growth, production
and resistance against cold and drought. Investigation on metabolic evaluation of seeds has shown that most
of them were hollow and early abscission. In this investigation, the effect of plant nutrition was studied
during 2 years in Gisoum region in Guilan province. The amount of potassium, calcium, natrium,
magnesium and phosphorus was measured by atomic absorption and spectrophotometer in leaves. Samplings
were done in four months (June, July, August and September). Sampling from soil was done and the
chemical and physical properties were determined. The amount of elements showed that the amount of Mg
was optimum but phosphorus was more and calcium was much more than required. In spite of optimum
amount of potassium in soil, measurement of K in leaves showed a severely deficient. Results indicated that
pH of soil has changed about 1- 2 unit from neutral to acidic (5-5.2) reaction during past 30 years. In acidic
soils, the absorption of K by roots is limited but the absorption of Ca is increased .This caused disorder in
Ca/K ratio. This situation along with climatical changes caused reduction in production and remaining of
seeds in ash.
Key words: Fraxinus excelsior -nutrient uptake- early abscission -seeds
Moraghebi, F* , Khanjanishirazi, B and Teimouri , M (2010) Relationship between some nutrient uptake and
early abscission of fruits in ash (Fraxinus excelsior ).Iranian J of Plant Physiology, 1(1): 37-42.

INTRODUCTION
Forest is a complex system according to ecological
definition and there is a balance between different
parameter quantitatively and qualitatively. By exact
understanding of these parameters, determining the
damaging factors and then preventing of its damage
would be possible.Fraxinus excelsior is a species
of Fraxinus native to most of Europe with the
exception of northern Scandinavia and southern
Iberia, and also southwestern Asia from northern
Turkey east to the Caucasus and Alborz mountains.
The resilience and rapid growth made it an
important resource for smallholders and farmers.
*Corresponding author :f.moraghebi@iausr.ac.ir

Received: August, 2010

Accepted: September, 2010
It was probably the most versatile wood in the
countryside with wide-ranging uses. Ash is one of
the most valuable and industrial species that grows
and regenerates rapidly in suitable condition such as
ecological and elite maternal stands and produces
seedlings with good quality. Sabeti studied the
distribution of Fraxinus excelsior in North of Iran
(Sabeti ,2001). The best sites of Fraxinus excelsior
are in north part of Iran (Tabari ,2002). This species
hasbeenusedwidely in aforestation and reforestation
plans because of its resistance against frost and
drought. The production of seeds has been
decreased because of forest damage. Most of the

seed are empty or early abscission (Korori and

Khoshnevis,2002).This situationwill cause extinction
of ash.
The nutritional demands of ash are high and
establish only in fertile soils. The occurrence
deficit of nutrients in ash is a signal that indicates
other species will suffer from deficit of nutrients
easily. The amount of elements in soil and their
optimum amount in leaves were studied in some of
forest trees including ash (Bonneaue, 1995). The
nutritional disorders were investigated in ash and
other species and determined the optimum amount
for suitable growth and regeneration (Tabari ,2002).
The deficiency of phosphorus in leaves was related
to deficiency of nitrogen and phosphorus in soil of
habitat (Zarrinkafsh, 1998). Investigation soil
fertility in Khirood Kenar forest showed deficiency
of all nutrient except for iron due to the composition
of soil bed (Mohammadi, 2001). The low growth of
Juniperus, Pistacia and Amygdalus was related to
deficiency of macro and microelements in soil of
Malek region in Kerman province (Ordoukhani ,
1998). The nutritional quality was lower in hazelnut natural forest in compare to artificial habitats
because of nutrient deficiency in soil of natural
habitats (Moraghebi, 2001). The optimum,
deficiency and excess level of different elements
was reported in some forest trees (Bonneaue, 1995).
The direct relationship was seen between deficiency
of elements in soil and Juniperus excelsa
(Espahbodi et al 2003). The effect of soil properties
in absorbance of Calcium by plants and antagonistic
effect on Fe and Mg caused early abscission of
seeds (Gennei, 1998).
The aims of this study were investigation of
nutritional situation in Fraxinus excelsior following
dramatic changes in Gisoum region due to wood
and pulp factory establishment, making road and
spreading of tourism in this region and show their
effect on seed production in this species.

MATERIAL AND METHODES

Study area
Selected trees (Figure 1) were located in Gisoum
region in Guilan province between 49o, 5 Eastern
longitude and 37o, 35 Northern latitude 10 m from
sea level. Its annual precipitation is about 1958 mm.
The minimum and maximum temperatures are 11
and 38/5 o C, respectively. Most of the soil bed is
composed of clay, volcanic stones and rarely sandy
rocks. The plant cover is 20% and the light percent
60% (according to plants crown).
Sampling

Sampling from leaves was done in June to

September in two successive years (2002-2003).
Samples were transferred to the laboratory at 4 C.
The percent of dry weight and organic and
inorganic compounds were determined. Leaves
were dried in 70 C for 48 hours and extracted by
dry ashing and use of HCl (2M) in 80 C for
measuring elements. The amount of calcium,
magnesium, natrium, potassium was measured by
atomic absorption spectrophotometer Phoenix 896
in extractions and by use of related standard
solution and calculated as g/100g. The amount of
phosphorous was measured by molibdate- vanadate
method and calculated as g/100g (Emami 2001).
Soil samples were taken from 0-20 cm depth and
analyzed according to standard methods.
Statistical analysis
Statistical analysis was done by T-test.

RESULTS
The height of ten selected Fraxinus individuals are
given in Table 1. As seen the height and diameter of
ranges between 12 to 25 m and 25-60 cm,
respectively. The percent of dry weight ranges from
35.2% to 46.1% and 31.2% to 41.6% in 2002 and
2003, respectively (Table 2) Results indicated a
decreasing amount of water from the beginning
until the end of season. As seen in table 2, the
amount of inorganic components showed a
increasing from June to August but with a little
decreasing in September.
The amounts of elements in 2002 and 2003 are
shown in Tables 3 and 4. During two years, the
amount of calcium was more than plant required.
The amount of magnesium and natrium was
optimum. The amount of Kalium has been changed
during different months but the comparison of its
amounts with standards showed a sever deficiency
in leaves. Also , there was a significant difference
between Kalium in two years. Results showed the
K/ Ca ratio was 0.36 and the ratio of K/Ca+Mg was
2.5-5 times less than normal ratio. The soil analysis
did not show K deficiency (Table 5). The reaction
of soil was acidic (pH 5-5.2).

DISCUSSION
Plants need 17 elements for normal growth. Carbon,
hydrogen, and oxygen come from the air and water.
Soil is the principle source of other nutrients.
Primary nutrients (nitrogen, phosphorus, and
potassium) are used in relatively large amounts by
plants, and often are supplemented as fertilizers.
Secondary nutrients (calcium, magnesium, and
sulfur) are also used in large amounts but are
typicallyreadilyavailableinadequate.

nutrient uptake and abscission relations in Ash 39

Micronutrients or trace elements are needed only in
small amounts. These include iron, zinc,
molybdenum, manganese, boron, copper, nickel,
and chlorine. In this study the amount of dry
weight, inorganic matter percentage, phosphore,
calcium, kalium, magnesium and natrium in leaves
was measured in different months in two
consecutive years. The pattern of changes in organic
matters and dry weight can be related to the efflux
of elements from leaves (Ebrahimzadeh, 2006;
Zarrinkafsh ,1998). According to Bonneau (Pvan ,
1993) sever deficiency in amount of K was seen in
all measured months in both years (Bonneaue ,
1995;Zarrinkafsh, 2001). The role of K in plant
physiology is very important and its deficiency
makes plants sensitive to weeds and disease
(Esphandiarpour ,2000). K deficiency along with
drought stress causes seeds early abscission
(Esphandiarpour ,2000; Ebrahimzadeh,2006, and
Haghparast 1999). The calcium is very critical in
plant nutrition and plants increase its uptake during
stressed condition to overcome it (Ebrahimzadeh,
2006, Bakrdjeva et al, 1996, Magholi ,1996; Pvan ,
1993). The K/C is another parameter that is
important in plant nutrition, flowering and yield
(Ebrahimzadeh, 2006, Haghparast, 1999). The
antagonistic relationship has been reported among
calcium, kalium and Magnesium uptake especially
Ca effects on K uptake (Ebrahimzadeh ,2006;
Haghparast, 1999; Salari ,2003;Pvan ,1993). The
ratio of K/Ca is 1/3 in a suitable condition.
According to the precipitation rate in July 2002 and
2003 (2mm and 16 mm respectively), the increased
uptake of calcium was predictable to resist against

drought stress. This condition caused abnormality in

K/Ca and K/Ca+Mg ratios and consequently a
decreasing in yield. The demand for kalium
increases during the stress. The flower has been hurt
as a result of late cold in Guilan during 2002-2004
and the rate of fecundated flowers has decreased
drastically. Sensitivity of trees against late cold can
be explained by two mechanisms: 1- The late cold
occurred after appearing of flowers. 2- The number
of buds decreased because of tree sensitivity against
late cold following K deficiency. Abnormality in
K/Ca and K/Ca+Mg caused a decreasing in yield
and losing of more seed during growth season. This
phenomenon intensified by occurring of drought in
June and July months. The acidic reaction of soil
limits the uptake of K and then its deficiency in
trees in spite of proper amount in soil (Tavallai
2001) The soil acidity changed from neutral 6/9 in
1975 to acidic 5 in 2002 because of different
factories activity (for example by wood factories).
Trees were cut for wood factory demand and then
this region was planted with nonendemic species
that caused drastic changes in plant cover and
ecosystem. Improving tourism, increasing the
population of region is another possible reasons for
ecosystem alteration. Local road has been changed
to highway with jam traffic. Making road in south
and west directions of this region interrupted in
natural drainage function. The level of underground
water will increase as a consequence of low
drainage, producing acidic humus. In acidic soil,
interruption in tree physiology and nutrient uptake
occur that finally cause reduction in seed
production.

Table1- Morphological parameter in selected trees

Tree number

Height (m)

Diameter (cm)

1
2

17
20

30
45

18

40

12

25

15

35

15

45

13

55

25

50

23

55

10

27

60

Table 2- Dry weight and inorganic matter percentage in leaves in different months.
June

July

August

September

%Dry weight (2002)

35.2

44

46.1

%Dry weight (2003)

31.2

32.7

35.9

41.6

%Inorganic matter (2002)

10

14.5

12.5

%Inorganic matter (2003)

8.2

8.26

10.74

10.08

Table 3- The average concentration of element in leaves of ash in 2002 (dry weight%)
P

Ca

July

0.354

1.09

.946

August

0.253

2.29

1.169

September

0.049

2.35

1.174

Table 4- The average concentration of element in leaves of ash in 2003 (dry weight%)
P

Ca

Mg

Na

June

2.65

1.489

0.725

0.235

0.06

July

0.724

1.63

0.601

0.227

0.065

August

0.684

2.09

0.72

0.226

0.071

September

0.506

1.65

0.71

0.222

0.072

Table5- Some chemical properties of soil

K

Na

Mg

Lime%

Ca

Cl

%OC

%N

%Chalk

373.52

83.02

9.12

0.78

46.4

0.66

4.12

0.346

32.92

K, Mg, Ca, P as mg/ kg soil

Cl as mg/g soil
trace

nutrient uptake and abscission relations in Ash 41

Figure 1- The Fraxinus excelsior site in Gisoum region in Gillan province

Figure2-Dispersion Fraxinus excelsior in Iran

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Bakedjeva, N.T., Christora, N.V., and Crstov,
K(1996) Reaction of peroxidase from different
plant species to in caved temperatures and the
effect of calcium and zinc ions , Plant peroxidase
IV international symposium. Geneva.
Bonneaue , M(1995) Fertilisation des forests dona
les pays temperes. Engerf, 324 pages.
Ebrahimzadeh ,H(2006) Plant physiology. Tehran
University publication, 230-252.
Emami,A (2001)Methods for plant analysis. Soil
and water research Institute publication,238-272.
Espahbodi, K , Mohannadnejad Kiasari, SH and
Barimaniuvarandi ,H( 2003) The best density
and mixed plantation of Acer and Fraxinus.
Forest and Populous research. 11:
19-34.
Esphandiarpour , P (2000) Investigation
relationship
between
physicochemical
properties of soil with plant covers in Juniperus
habitats in Malek region in Kerman province.
Islamic Azad University Publication, 357 pages.
Gennei, E. , Bussotti,F.,and Galeotti, L (1998)
The declune of Pinus nigra . Reforestation
stands on limetone substructure. Ann. Sci. For .
Elsevier.
Haghparast,M (1999) Plant physiology.Gillian
University Publication,412 pages..
Korori, S.A.A., Khoshnevis,M (2002) Metabolic
evaluation of Fraxinus seeds by use of enzymes
and cations alterations. Journal of Forest and
Populous research. 9: 83-149.
Magholi, F(1996) Physiological reasons of
yellowing in natural and artificial habitats of
Haloxylon sp. Pajouhesh Va Sazandegi in
Natural Resources Journal 53: 21-30.

Mohammadi, F(2001) Investigation of trees

nutrition and fertility of different ecosystems in
Khiroodkenar
forest.Tehran
University
Publications, 150-165.
Moraghebi, F(2001) Ecological studies and
environmental adoption and plant sociology in
hazel-nut sites in north of Iran, Pajouhesh Va
Ssazandegi in Natural Resources Journal,51: 110.
Ordoukhani, K(1998) Ecophyiological studies of
Juniperus, Pictacia and Amygdalus forest in
Malek region in Kerman province. Islamic Azad
University Publication, 355 pages.
Pvan , X (1993) Association of calcium and
calmodulin to peroxidase secretion and activation
. J Plant Physiology . 141: 141-146.
Sabeti ,H(2001)Iranian trees and shrubs. Iranian
Botanical Garden publication.890 pages.
Salari, A.A (2003) Plant nutrition. University
Publication Center,412 pages.
Tabari,
M
(2002)Forest
population
and
environmental requirement of Fraxnius excelsior
in north of Iran. Pajuhesh va Sazandegi . 55: 94103.
Tavallai, M (2001) Hydroponics cultures in
commercial scale. Publication of agricultural
press. 530 pages.
Zarrinkafsh ,M (1998) Physico-chemical properties
of soil in Lajim (Zarab), Agriculture faculty.
Tehran University press, 214 ,pages,
Zarrinkafsh ,M(2001) Forest soils. Research
Institute of Forests and Rangelands press, 349
pages.

Effects of Nitrate on Nodulation and Important

Element Contents in Inoculated Phaseolus vulgaris L.
Niloufar Shoarian, Kamaleddin Dilmaghani* and Hasan. Hekmatshoar
Department of Biology ,Islamic Azad University Marand Branch , Marand, Iran

ABSTRACT
In this investigation, the effects of different levels of nitrate on nodulation inoculated root system of
Phaseolus vulgaris L. (Shiny red) as well as dry weight of root, stem and leaves and also content and
distribution of Na+, K+, Ca++, Mg++ and phosphorus in the organs of treated plants have been studied. The
obtained results showed that low amount of nitrate increased dry weight of root, shoot and leaves. In
contrast, high amount of nitrate at10 and 15mM decreased above mentioned growth parameters. Increased
nitrate levels caused an increase of K +, Na+, Mg++ and Ca++ content of root, stem and leaves. The Na+, Ca++
and Mg++ contents in leaves and K+ contents in root system of plants were considerably higher than their
content in other organs. Phosphorus content of different organs of plants also showed an increase when
nitrate levels increased. The presence of leghemoglobin in nodules was considered as an index for its
nitrogen fixation activity. The size and number of nodule decreased with increasing the nitrate levels. High
level of nitrate at15mM completely inhibited nodulation processes.
Key words: Phaseolus vulgaris, nitrate effects, nodulation, growth parameters, cation and phosphorus
content
Shoarian , N , Dilmaghani, K* and Hekmatshoar , H (2010) Effects of Nitrate on Nodulation and Important
Element Contents in Inoculated Phaseolus vulgaris L.Iranian J of Plant Physiology, 1(1): 43.47.

INTRODUCTION
Nitrogen is a major element for all living
organisms. The use of NO 3 from soil or fertilizer
and N2 by symbiotic association with rhizobia,
simultaneously or in a complementary way by
nodulated legumes, is a unique characteristic among
higher plants (Becana and Sprent, 1987). Legumes
are very important both ecologically and
agriculturally because they are responsible for a
substantial part of the global flux of nitrogen from
atmospheric N2 to fixed forms such as ammonia,
nitrate, and organic nitrogen (Brockwell et al,
1995).
Nitrate constitutes the major source of nitrogen
in the great majority of aerated cultivated soils. At
high concentration, nitrate inhibits both nodulation
and N2 fixation in almost all legume species
(Arrese-Igor et al, 1997). However, a low
Corresponding author : k0_dil@yahoo.com
Received: June, 2010
Accepted: September, 2010

concentration of nitrate favours the initial

establishment of the nodulated plant, perhaps as an
additional source of nitrogen (Becana and Sprent,
1987). In this study, we showed the effect of various
levels of nitrate on the growth and Na+, K+, Ca++,
Mg++ and phosphorus content in the organs, nodule
density and size.

MATERIALS AND METHOD

The seed of Phaseolus vulgaris Shiny red was
provided from Seed and Tree improvement Center,
Karaj-Iran. Rhizobium was obtained from the
nodules of the plant previously affected by this
bacterium, then cultured in nutrient-agar medium.
The bacteria added to the clay soils were used to
inoculate. Inoculated seedlings were transferred into
plastic pots, one plant per pot. Plants were grown in
soil with texture of sandy-loam under following
conditions: light intensity 10000 lux, light and dark
period, 12-12h, temperature, 25 5 C, soil pH

7. .The plants were treated with, 1, 2.5, 5, 10 and

15mM of nitrate. Six weeks old plants were
harvested and important growth parameters and also
content of Ca2+and Mg2+ by complexometry Na+and
K+ by flame photometry and phosphorus of the
organs were recorded.

RESULT
The results presented in Table 1 shows the growth
parameters of the plants supplied with 1, 2.5 and

5mM of NO3- increased with increasing nitrate, in

the concentration greater than 5mM such as 10 and
15mM of NO3- the amount of biomass decreased
significantly. Increasing NO3- concentrations
decreased size and number of nodules, in 15mM of
NO3- inhibited nodulation (Table2&3). Increased
nitrate levels caused an increase of Ca2+, Mg2+, Na+,
K+ in the organs. Phosphorus content also showed
an increase.

Table 1.Effect of different levels of NO3- on dry weight of stem, leaf and root of Phaseolus vulgaris inoculated and
non-inoculated with rhizobium

TREATMENT

[NO3-]Rhizobium

Leaf dry weight

Root dry weight

Control 1

1mM - Rh.

0.46a 0.02

0.11a 0.02

0.25a

Control 2

Rh.+ 1mM

0.46a 0.06

0.11a 0.01

0.27a 0.03

Treatment 1

.Rh+2.5mM

0.47a 0.02

0.13a 0.03

0.27a 0.02

Treatment 2

Rh.

+ 5mM

0.5a 0.06

0.14a 0.04

0.26a 0.01

Treatment 3

Rh.

+10mM

0.49a 0.02

0.15a 0.03

0.25a 0.00

0.29b 0.02

0.07b 0.01

0.14b 0.01

Treatment 4

Rh.+ 15mM

Difference between averages presented in each column having common letter are not significant at p<0.05
Table 2. Effect of different levels of NO3- on the averages of number and of diameter
nodules of Phaseolus vulgaris inoculated and non-inoculated with rhizobium
Treatment

[NO3-]Rhizobium

Nodule diameter

Nodule number

Control 1

1mM - Rh.

0.00d 0.00

0.00d 0.00

Control 2

Rh.+ 1mM

2.49a 0.3

23.00a 3.00

Treatment 1

.Rh+2.5mM

1.39b 0.21

18.33b 4.04

Treatment 2

Rh.

+ 5mM

1.02c 0.15

6.67c 2.89

Treatment 3

Rh. +10mM

0.73c 0.14

3.33cd 1.15

Treatment 4

Rh.+ 15mM

0.00d 0.00

0.00d 0.00

Nodulation study in inoculated Phaseolus vulgaris

45

Difference between averages presented in each column having common letter are not significant at p=0.05
Table 3. Effect of different levels of NO3 on the averages of Ca2+ content in root, stem and leaf of
Phaseolus vulgaris inoculated and non-inoculated with rhizobium
Treatment

[NO3-]Rhizobium

Root

Stem

Leaf

Control 1

1mM - Rh.

22.03a 1.95

16.58a 2.26

17.07b 1.29

Control 2

Rh.+ 1mM

22.42a 2.65

18.87a 1.83

17.85b 2.89

Treatment 1

.Rh +2.5mM

24.04b 3.51

19.85a 1.15

17.92b 2.46

Treatment 2

Rh.

+ 5mM

25.22b 1.34

18.96a 4.38

19.73ab 0.01

Treatment 3

Rh.

+ 10mM

25.05b 2.42

20.59a 2.07

21.45a 1.09

33.17c 1.61

23.33a 2.97

23.03a 0.54

Treatment 4

Rh.+ 15mM

Difference between averages presented in each column having common letter are not significant at p=0.05
Table 4. Effect of different levels of NO3 - on the averages of Mg2+ content in root,stem and leaf of
Phaseolus vulgaris inoculated and non-inoculated with rhizobium
Treatment

[NO3-]Rhizobium

Root

Stem

Leaf

Control 1

1mM - Rh.

24.22b 3.43

16.68b 1.11

9.93b 0.55

Control 2

Rh.+ 1mM

26.08b 4.93

17.04b 3.48

10.99b 0.8

Treatment 1

.Rh +2.5mM

36.64ab 1.82

18.45b 5.57

13.38b 1.13

Treatment 2

Rh.

+ 5mM

42.6ab 3.67

20.47b 4.48

14.72ab 2.62

Treatment 3

Rh.

+ 10mM

44.39a 7.3

22.27b 6.2

16.1a 4.3

50.6a 6.92

26.26a 4.48

18.33a 4.91

Treatment 4

Rh.+ 15mM

Difference between averages presented in each column having common letter are not significant at p=0.05
Table 5. Effect of different levels of NO3 on the averages of Na+ content in root, stem and leaf of
Phaseolus vulgaris inoculated and non-inoculated with rhizobium
Treatment

[NO3-]Rhizobium

Root

Stem

Leaf

Control 1

1mM - Rh.

6.46b 1.14

2.1b 0.44

0.36a 0.05

Control 2

Rh.+ 1mM

7.1b 1.13

2.38b 0.22

0.46a 0.07

Treatment 1

2.5mM+Rh.

8.98a 1.2

2.5b 0.47

0.47a 0.06

Treatment 2

Rh.

+ 5mM

9.91a 0.59

2.61b 0.53

0.89a 0.05

Treatment 3

Rh.

+10mM

10.1a 1.1

2.96ab 0.9

0.98a 0.36

11.3a 1.12

3.38a 0.378

1.29a 0.42

Treatment 4

Rh.+ 15mM

Difference between averages presented in each column having common letter are not significant at p=0.05

Table 6 . Effect of different levels of NO3 on the averages of K+ content in root, stem and leaf of
Phaseolus vulgaris inoculated and non-inoculated with rhizobium
Treatment

[NO3-]Rhizobium

Root

Stem

Leaf

Control 1

1mM - Rh.

10.69 b 1.65

27.3b 2.37

23.32b 2.38

Control 2

Rh.+ 1mM

13.4b 2.88

27.6b 2.87

24.39b 2.7

Treatment 1

.Rh+2.5mM

20.31a 1.15

31.34a 2.03

25.58b 2.26

Treatment 2

Rh.

+ 5mM

21.1a 2.95

31.06a 2.74

26.27ab 1.77

Treatment 3

Rh.

+ 10mM

22.23a 1.87

31.38a 1.76

34.49ab 3.14

22.97a 1.23

32.2a 2.85

35.04a 2.61

Treatment 4

Rh.+ 15mM

Difference between averages presented in each column having common letter are not significant at p=0.05
Table 7. Table 6 . Effect of different levels of NO 3 on the averages of phosphorus content in root,
stem and leaf of Phaseolus vulgaris inoculated and non-inoculated with rhizobium
Treatment

[NO3-]Rhizobium

Root

Stem

Leaf

Control 1

1mM - Rh.

1.02d 0.12

0.6b 0.28

0.27b 0.05

Control 2

Rh.+ 1mM

1.23d 0.48

0.63b 0.3

0.45b 0.13

Treatment 1

2.5mM + Rh.

2.74cd 0.47

0.78b 0.05

0.69a 0.11

Treatment 2

Rh.

+ 5mM

4.15c 0.76

0.91a 0.14

0.74a 0.12

Treatment 3

Rh.

+ 10mM

6.48b 0.63

0.96a 0.07

0.88a 0.1

8.96a 1.30

1.15a 0.07

0.93a 0.21

Treatment 4

Rh.+ 15mM

Difference between averages presented in each column having common letter are not significant at p=0.05

DISCUSSION
The above reported results, make possible to have a
brief comment as on the above presented results
follow: high amount of dry weight-biomass- in the
studied genotype, Phaseolus vulgaris, and its low
amount in plants inoculated with rhizobium and
treated with 10 and 15mM of nitrate agreed with
results of Andrews et al (1990) and Silveira et al
(2001), and could be interpreted by lowing size,
density and the rate of nodulation in plants grown in
soil containing high level of nitrate. Nodulation

suppressed in the concentration above 10mM of

NO3-(Streeter 1984, Lucinsky et al. 2002, Silveira
2001). Increasing nitrate concentration in root
medium increased content of Ca2+, Mg2+and Na+ in
root, stem and leaf. Content of these elements in
root is higher than their content in stem leaf. K+
content increased by the increasing of nitrate levels
in root medium. The ratio of leaf K+ content to that
of stem and root was higher than 2. It appears that
nitrate enhanced K+ transport from root to shoot.

Nodulation study in inoculated Phaseolus vulgaris

The same results were reported by Carolus 1988,

Yanai et al 1996 and Rains 2005. Contrary to
results of most research work, in our study the
content of phosphorus increased in plants in the
medium containing high level of NO3- the highest
content of phosphorus belonged to leaves and
lowest content of it belonged to stem the similar
results presented previously by Metwally et al
(2007).

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activity in the leguminosae. Phytochemistry
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Arrese-Igor, C.,Minchin, F.R., Gordon, A.J.,
and Nath, A.K (1997) Possible causes of the
physiological decline in soybean nitrogen
fixation in the presence of nitrate. Exp Bot, 48:
905-913.
Becana, M., and Sprent, J (1987) Nitrogen
fixation and nitrate reduction in the root nodules
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Manipulation of rhizobia microflora for
improving legume productivity and soil fertility:
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Carolus, Robert L(1938) Effect of certain ions,
used singly and in combination, on the growth

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and potassium, calcium, and magnesium

absorption of the bean plants. Plant
Physiol13(2):349-363.
Lucinski, R., Polcyn, W., and Ratajczak,
L(2002)Nitrate reduction and nitrogen fixation in
symbiotic association Rhizobium-legumes. Acta
Biochimica Polonica, 49( 2):321-327.
Metwally, AL.,El-Damaty, M., and Mustafa,
M(2007)Salt influence on nitrate and phosphate
uptake by barley in sand culture, Zeits chrift fur
Pflan zenernahrung und Bodenk unde, 141(4):
411-418.
Rains, D.W (2005) Cation absorption by slices of
stem tissue of bean and cotton. Cellular and
Molecular Life sciences (CMLS), 25(2): 512-216.
Silveria,J.A.G.,Matos, J.C.S.,Ceccato, V . M .,
Viegas, A. and Oliveira, J.T.A(2001) Nitrate
redutase activity, distribution, and response to
nitrate in two contrasting phaseolus species
inoculated with Rhizobium spp. Environmental
Botany, 46(1): 37-46.
Streeter, J.G(1985) Nitrate inhibition of legume
nodule growth and activity, Plant Physiol, 77:
321-324
Yanai, J.,Linehan Denis, J.,Robinson, D.,Young
Iain, M.,Hackett Christine, A., Kyma, K.,
and Kosaki, T(1996) Effects of inorganic
nitrogen application on the dynamics of the soil
solution composition in the root zone of maize.
Plant and Soil, 180(1): 1-9.

48

Short Communication
Antimicrobial Activity of Crude Extracts taken from In
vitro and In vivo grown Ocmium basilicum L.
Muafia shafique , Shaista Jabeen Khan* and Nuzhat Habib Khan
Food and Biotchnology research Centre, Pakistan Council of Scientific and Industrial
Research Laboratories Complex, Lahore, Pakistan

ABSTRACT
The antimicrobial activities of in vitro grown callus extract and in vivo grown Ocimum basilicum L. plant
leaves extracts were studied and compared. Effect of extraction solvent was also assessed. These extracts
were tested in vitro against eight bacterial strains following disc diffusion method. The results indicated that
in vitro grown callus extracts of O. basilucum exhibited higher antimicrobial activity against tested Gram
positive microorganisms as compared to in vivo grown plant material extract. These findings indicate
towards potential use of biotechnology for natural therapeutic agent production.
Key words: O. basilicum, antimicrobial activity, tissue culture, medicinal plant
Muafia S., Shaista J. Khan* and Nuzhat Habib Khan(2010) Antimicrobial Activity of Crude Extracts taken
from In vitro and In vivo grown Ocmium basilicum L. Iranian J of Plant Physiology, 1(1): 48 - 52 .

INTRODUCTION
Multiple drug resistant pathogenic microorganisms
affecting both human and plant are developed due
to the arbitrary use of commercial antibiotics in the
treatment of infectious diseases (Kalemba, and
Kunicka, 2003). Scientific community is now
paying attention to find efficient plants against
microbial growth (Yayasinghe et al, 2003). O.
basilicum (family Lamiaceace), commonly called
Sweet Basil is known as king of herbs which
contains plenty of phytochemicals with significant
nutritional as well as antioxidant capabilities and
health benefits (Nyak and Uma ,2005 ). Sweet Basil
has shown unique health protecting effects due to its
important flavonoids and volatile oils (Adiguzell et
al ,2005). A plenty of work has been done on sweet
basil regarding its anti microbial properties.
However, in vitro grown Ocimum basilicum callus
extract has not been reported in terms of
antimicrobial activity.
* Corresponding author: Dr_shaista13@hotmail.com
Received: July, 2010
Accepted: September, 2010

The purpose of this study was to compare the

potential antimicrobial activity of in vitro grown
callus with that of in vivo growing O . basilicum
plants.

MATERIAL AND METHODS

Sweet basil (Ocimum basilicum L.) grown in the
garden of PCSIR Laboratories Complex, Lahore
were used during present study. The surface
sterilized leaves were cut in to 0.5-1cm small pieces
under aseptic conditions and inoculated on sterilized
full strength MS medium (Murashige and Skoog,
1962) supplemented with different dosages of 6Benzyl amino-purine (BA) in combination with
1.07M Nephthalene acetic acid (NAA). BA was
supplied from 0.44 M to 8.88M. After
inoculation these cultures were kept for 21 days in a
growth chamber maintained at 252C under 16h
photoperiod and 48mol m-2 s-1 light intensity for
callogenesis. Four replicates per treatment were
used.
To prepare plant extracts absolute methanol
(99%) and 70% aqueous methanol were used as
solvents. Four kinds of plant extracts were prepared

which included in vitro grown callus extract (21

days old callus) and in vivo grown Ocimum
basilicumL.plants leaves extracted both with
absolute methanol and 70% aqueous methanol
separately.
In vitro antimicrobial studies were carried out on
eight bacterial strains (Escherichia coli ATCC
25922, Bacillus subtilis ATCC 6633, Klebsiella
pneumoniae, Salmonella paratyphi , Pseudomonas
aeruginosa, Staphylococcus aureus, Escherichia coli
and Enterobacter species) maintained as stock
cultures in their appropriate agar slants at 4C. For
antibacterial assay 24h old bacterial cultures at
371 C were used. Cultures were diluted 10-1 in
sterile ringer solution (Collins, 1967) containing
approximately 106CFU/mL in each case. Twenty
five micro-liters of these suspensions were
inoculated over plates containing sterile nutrient
agar medium using a sterile cotton swab in order to
get a uniform microbial growth on both control and
test plates. Paper disc diffusion method (Bauer et.al,
1966) was applied with slight modification to test
the antimicrobial activity. Filter paper discs each
impregnated with 30L of each plant extract were
placed on pre-inoculated culture media and
incubated at 371C for 24h. All experiments were
performed in duplicate. Penicillin, co-trimoxazole
and Streptomycin were used as positive controls.
25L of each antibiotic solution (0.01g/10mL) was
dropped on paper discs. 25L of 10% aqueous
solution of dimethylsulfoxide (DMSO) was used as
negative control during this study.

RESULTS AND DISCUSSION

Effect of different concentrations of BA (0.448.88M) in combination with NAA (1.07 M) on
callus induction in leaf explants of Ocimum
basilicum L. is shown in Table-1. Explants
responded to all concentrations of BA along with
NAA producing calli having different attributes.
Superior sweet basil calli were produced on MS
medium supplemented with BA (8.88M) along

with NAA (1.07 M) in terms of callus induction

(100% explants). Whereas lesser quantity of BA
(0.88M) in the presence of 1.07 M NAA was
found to be least effective for callus induction only
37.5% cultures showed callus induction as shown in
table-1.
The antimicrobial activities of O. basilicum in
both in vitro callus and in vivo leaf extracts
(absolute or 70% aqueous methanol) against eight
microorganisms were examined in the present study,
the results are shown in table-2. It was observed that
both extracts of in vitro grown callus were effective
against two microorganisms B. subtilis ATCC 6633
and S. aureus whereas in vivo grown leaf extracts
were effective against only B. subtilis ATCC6633 as
shown in table-2. All other bacterial strains were
resistant to these extracts. These results depicted
that in vitro callus extracts showed better
antimicrobial properties as compared to in vivo
grown leaves extract.
Effect of various solvents used for extraction on
their antimicrobial properties of in vivo grown
Ocimum basilicum plants was studied by Kaya et al.
(2008) and Adiguzel et al. (2005). Whereas present
results report for both in vivo as well as in vitro
grown O. basilicum plants. Streptomycin was
effective against both Gram positive and Gram
negative microbs whereas penicillin was effective
against only Gram positive microorganisms. On the
other hand all microorganisms investigated were
resistant to Co-trimoxazole.The results are shown in
table-2.
The striking finding of this investigation is that
B. subtilis and S. aureus found to be resistant to
standard antibiotic co-trimoxazol were sensitive to
in vivo grown callus extract of Ocimum basilicum
L. Consequently the extracts of in vitro grown callus
have larger antimicrobial potential as compared to
in vivo grown leaf extracts of O. basilicum These
finding indicate the potential use of Biotechnology
for natural bioactive material production from sweet
basil using appropriate procedure/ technology.

Table-1. Effect of NAA in combination with BA on callogenesis in Ocimum basilicum leaf explants.
Medium
composition

Sr.#

(M)

MS+1.07NAA
1

Callus
initiation
%

Callus
growth

Morphogenetic
potential

Remarks

40

+++

Root initiation

Callus Compact green

38

Nil

Callus Light green &loose

70

+++

Nil

Callus some what

yellowish vitrifaction

50

++

Root initiation

Callus Compact whitish

green

100

++++

Nil

Callus Compact granular

light green with powdery
upper surface

+0.44BA

MS+1.07NAA

+0.88BA

MS+1.07NAA

+2.22BA

MS+1.07NAA

+4.44 BA

MS+1.07NAA

+8.88 BA
+

= Poor

++

= Good

+++

= Very Good

+ + + + = Excellent

Table-2. Assessment of antimicrobial activity in four different extracts of O. basilicum against eight
microorganisms
Microorganisms

Zone of inhibition (mm)

Streptomycin

Penicillin

Cotrimoxazole

25g/disc

25g/disc

25g/disc

21

8.5

28

16.5

18.5

14

22

Staphylococcus
aureus HI

8.0

7.5

19

26

Escherichia coli
HI

24

Enterobacter
species HI

22

MCE

ACE

MLE

ALE

DMS

Escherichia coli
ATCC 25922

Bacillus subtilis
ATCC 6633

12.5

11.5

9.0

Klebsiella
pneumoniae HI

Salmonella
paratyphi HI

Pseudomonas
aeruginosa HI

Absolute Methanol callus Extract

= MCE
70% aqueous Methanol callus extract = ACE
Absolute Methanol Leaves extract
= MLE
70% aqueous Methanol Leaves extract = ALE
Hospital isolated pathogen
=HI
No inhibition zone (resistant)
= (- )

REFERENCES
Adiguzell, A., Glluce, M., Sengul, M., Ogutcu, H.,
Sahin, F., and Karaman, I (2005)
Antimicrobial Effects of Ocimum basilicum
(Labiatae) Extract. Turkish Journal of Biology,
29, 155-160.
Bauer, A.W., Kirby, M.D.K., Sherris, J.C., and
Turck, M (1966) Antibiotic susceptibility testing
by standard single disc diffusion method.
American Journal of Clinical Pathology, 45, 493496.
Collins, C.H (1967) Microbiological methods
London: Butterworths.
Kalemba, D.,and Kunicka,A (2003) Antimicrobial
and antifungal properties of essential oils.
Current Medicinal Chemistry, 10, 813 829.
Kaya, I., Yigit, N.,& Benli, M (2008)
Antimicrobial activity of various extracts of
Ocimum basilicum L. and observation of the

inhibition effect on bacterial cells by use of

Scanning Electron Microscopy. African J.
Traditional Complemen. and Alterna. Med. 5,
363 369.
Murashige, T., and Skoog, F(1962) A revised
medium for rapid growth and bioassays with
tobacco tissue cultures. Physiologia Plantarum,
15, 473497.
Nyak, V., and Uma, D.P (2005) Protection of
mouse bone marrow against radiation-induced
chromosome damage and stem cell death by
Ocimum flavonoids Orientin and vicenin.
Radiation Research, 163(2), 165-171.
Yayasinghe, C., Gotoh, N., Aoki, T., and Wada S
(2003) Phenolics composition and antioxidant
activity of sweet basil (Ocimum basilicum L.).
Journal of Agricultural and Food Chemistry, 51,
4442-4449.

53

Troubleshooting with you

DNA extraction
Fatima Shahhosseini*
PhD student, Genetic and Molecular Biology, Faculty of Science, University of Malaya, Kuala Lumpur,
Malaysia
*fatima3132002@yahoo.com

What is DNA Extraction?

Simply put, DNA Extraction is the removal of deoxyribonucleic acid (DNA) from the cells in which it
normally resides. The research for a more efficient means of extracting DNA of both higher quality and yield
has lead to the development of a variety of protocols, however the fundamentals of DNA extraction remains
the same. DNA must be purified from cellular material in a manner that prevents degradation.

What is it used for?

Extraction of DNA is often an early step to diagnose many medical conditions and can also be used for
genetic engineering of both plants and animals such as FISH, PCR, RFLP, and Sequencing. It can also be
used to gather evidence in a crime investigation.

How does it work?

Here is the outline of a basic DNA Extraction:
First step is to lyse the cells containing the DNA of interest by grinding with Extraction buffer, secondly
DNA associated proteins, as well as other cellular proteins, may be degraded with the addition of a protease.
DNA later is the precipitated by mixing with cold ethanol or isopropanol and then centrifuging. Wash the
resultant DNA pellet with cold alcohol again and centrifuge for retrieval of the pellet is the last step. After
pouring the alcohol off the pellet and drying, the DNA can be re-suspended in a buffer such as Tris or TE.
Presence of DNA can be confirmed by electrophoresing on an agarose gel containing EtBr, or another
fluorescent dye that reacts with the DNA, and checking under UV light. When DNA extractions are
performed, you can expect three basic results.
1. No DNA which either cell was not lysed enough or DNA was lost during the experiment.
2. DNA appears fluffy which means it has sheared in the extraction process.
3. DNA appears as thin threads which shows genomic DNA was extracted.
Regardless which protocol is being used to extract DNA, here is some tips:
Do not allow the pellet of DNA to dry completely as it makes it very difficult to dissolve.
If Phenol is being used, make sure pH of the Phenol is ~8.0 to prevent DNA from becoming trapped at the
interface between the organic and aqueous phases.
Always cool the mortar slowly by adding small amount of liquid nitrogen over a period of time. Sudden
immerse the grinding part of the pestle in liquid nitrogen can cause fracturing.
If DNA of higher molecular weight is required, take care to minimize shearing forces and do not vortex
vigorously; but bear in mind that vortexing ensures a greater yield of DNA composed of fragments up to
20kb in length that can be detected by southern hybridization, dot and slot blotting and PCR analysis.
If possible, it is preferable to collect young tissues since they have more cells per weight and therefore
result in higher yields. In addition, young leaves have not accumulated as much polysaccharide,
polyphenolics and secondary metabolites which inhibits restriction enzymes as well as other DNA modifying
enzymes.
After harvesting, if plant tissue will not be used immediately, it should be frozen in liquid nitrogen. It can
then be stored at 80C for later processing. Ground tissue powder can also be stored at 80C.

If your lysate is too viscous, reduce the amount of starting material or increase the amount of
extraction buffer like CTAB buffer.

Abbreviation
TE buffer: Tris-Cl. EDTA
FISH: Fluorescence In Situ Hybridization
RFLP: Restriction Fragment Length Polymorphism
EtBr: Ethidium Bromide
CTAB: CylTrimethylAmmonium Bromide
PCR: Polymerase Chain Reaction

Read more on
Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor
Laboratory Press Sambrook and Russell
http://serc.carleton.edu
http://www.accessexcellence.org
http://www.protocol-online.org
http://www.ambion.com/techlib

55

BOOK REVIEW
Ghorbanli , M. and Bonyadi, R (2008) Advanced Plant Metabolism , Islamic Azad University Gorgan Branch
Press. ISBN: 978-964-450-942-1. 496 pp.

The first edition of this successful publication of the well known professor of scientific writing gives me
an opportunity to introduce it to all of plant physiology interesting peoples. This exciting new book provides
an up-to-date survey of the biochemistry and physiology of plant metabolism. The proof commences with an
overview of the biochemistry, physiology and function of primary and secondary metabolism, followed by
detailed reviews of the major concepts of photosynthesis and respiration. This book has 5 chapters discusses
the energetic concept of metabolism, photosynthesis, the apparatus of photosynthesis, light and dark
photosynthetic reactions and cellular respiration. Completely brings right up to date with much new
information, this book is an essential purchase for advanced students, researchers and professionals in
biochemistry, physiology, molecular biology, genetics, plant sciences, agriculture, working in the academic
sectors. Libraries in all universities and research establishments where these subjects are studied and taught
will need copies of this excellent book on their shelves.
But generally, this is one of the best books on plant physiology: buy it!
Mozhgan Farzami Sepehr

IJPP
Iranian Journal of Plant Physiology
Managing Editor:
Mozhgan Farzami Sepehr(PhD)
Assistant Professor
Department of Biology
Faculty of Agriculture
Islamic Azad University Saveh Branch
Saveh, Iran

farzamisepehr@iau-saveh.ac.ir

(www.aup.edu.pk)
drihkhalil@gmail.com

Jennifer Ann Harikrishna (PhD)

Associate Professor,
Genetics and Molecular Biology
Institute of Biological Sciences
Faculty of Science
University of Malaya
50603 Kuala Lumpur
Malaysia

jennihari@um.edu.my

Editor in Chief:
Mahlagha Ghorbanli(PhD)

Gholamreza Bakhshi Khaniki (PhD)

Professor
Department of Biology
Faculty of Science
Islamic Azad University Gorgan Branch
Gorgan, Iran

Professor
Faculty of Agriculture
Payame Noor University
Lashkarak Road
Tehran , Iran
P.O.Box:14335-333

ghorbanli@yahoo.com

Executive Editor:
Mona Farhadi(PhD)
Assistant Professor
Department of Biology
Faculty of Agriculture
Islamic Azad University Saveh Branch
Saveh, Iran

monafarhadi@yahoo.com

Editorial Board:
Iftikhar Hussain Khalil (PhD)
Professor
Plant Breeding and Genetics Department
NWFP Agricultural University
Peshawar, Pakistan

Fariba Meighani(PhD)
Assistant Professor
Iranian Research Institute of Plant
Protection

fmaighany@yahoo.com

Maryam Shahbazi (PhD)

Assistant Professor
Molecular physiology Department,
Agriculture Biotechnology Research Institute of
Iran (ABRII)
Mahdasht Road
P. O. Box 31535-1897
Karaj, Iran

MShahbazi@abrii.ac.ir
http://www.abrii.ac.ir

BOOK REVTEW

57

published before and is not under consideration for

publication anywhere else.

Seyed Mohammad Mahdi Hamdi (PhD)

Assistant Professor
Dep. of Biology
Faculty of Science
Islamic Azad University of Garmsar

Garmsar, Iran
mm_hamdi@asia.com

Mohamad Ali Baghestani Meibodi(PhD)

Associate Professor
Iranian Research Institue of Plant Protection

baghestani40@hotmail.com

Iranian Journal of Plant Physiology is a quarterly

journal published by Islamic Azad University
Saveh Branch in English. Manuscripts may be
submitted in English. Tables of contents and other
useful information, including these instructions for
contributors, are available at the websites of the
Islamic Azad University Saveh Branch and the
Editorial Office (Department of Biology, Faculty
of Agriculture, and Islamic Azad University Saveh
Branch.

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This journal publishes the new results of
completed, original studies on any aspect of plant
physiology based also on approaches and methods
of biochemistry, biophysics, genetics, molecular
biology, genetic engineering, applied plant
physiology, and other related fields. We also accept
descriptions of original methods and instruments
opening novel possibilities for obtaining and
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All papers, including Brief communications,
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Within the text (INTRODUCTION, MATERIALS
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Journal articles should be cited as follows:

McDougall, G.J., Stewart, D., and Morrison,
I.M (1994) Cell-Wall-Bound Oxidases from
Tobacco (Nicotiana tabacum L.) Xylem Participate
in Lignin Formation, Planta, 194: 914.
For correct abbreviations of journal titles, refer to
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Books should be cited as follows:
Cobb, A (1992) Herbicides and Plant
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follows:

BOOK REVTEW

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Lichtenthaler, H(1996) Vegetation Stress: An

Introduction to the Stress Concept in Plants,

permitted. Load your figures at 600 dpi (dots per

inch) for linear

Vegetation Stress, Lichtenthaler, H., Ed.,

Stuttgart: Gustav Fisher, 414.

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Editorial processing
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and Zinc Ions on the Meristem Cell Arrangement
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(Biol.) Dissertation, Moscow: Mosk. Gos. Univ.
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Islamic Azad University Saveh Branch Publisher

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in the research project should have contributed to the conception and design of the project, drafted
substantive portions of the paper and taken responsibility for the analysis and conclusions of the paper. Other
participants with less responsibility should be identified and acknowledged for their contributions.

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