1

Gibberellic acid and Cold Effect on Protein Variation in Ferula gummosa Boiss. seeds

Shirin Haddad Kaveh*, Zeinab Pasban Vatan and Françoise BernardPlant Physiology and Biotechnology Laboratory, Faculty of Biological Sciences University of Shahid

Beheshti ,Tehran, Iran

ABSTRACTSeeds 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 (-20°C, 4°C, 8°C) 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 GA3 or 4°C 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 4°C 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 .

INTRODUCTIONFerula 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.comTel : +98-912-7040115Received: July, 2010Accepted: 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

2 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 METHODSeed Collection

Galbanum seeds were collected from Firouzkouh Mountains (Iran) in September 2008 and they were kept in 4°C until used for the experiment.

Germination in different treatmentsFirstly 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 and then transferred to 1%(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 4°C. The second group was kept at -20 °C for 2 weeks and then transferred to 4 °C, and the third group was stored at 4°C for 2 weeks and then transferred to 8 °C. Petri dishes were sealed using parafilm.

Protein AnalysisAn 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 protein analysis was performed according to Bradford

(1976) test and electrophoresis for qualitative measurements of proteins was performed by SDS-PAGE. Alpha-Amylase marker (Merck) was also used as a standard.

Statistical AnalysisAll data in this study were statistically analyzed

using LSD test and analysis of variance (ANOVA) by SPSS 11 software.

RESULTS Ferula gummosa Seed GerminationThe 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 4°C 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 4°C cold treatment ( see table 1).The percentage of germination in other treatments for temperature was very low nearly zero.

Qualitative Protein AnalysisNon 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 4°C. 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 4°C for breaking dormancy, one may 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 3

in germinated seeds treated by 1000mM GA3 and 4°C. 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 α-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 4°C 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, GA3

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 GA3

and coldness or in a lesser extent under the effect of cold treatment at 4°C 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 4°C 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 4°C 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 alpha-amylase 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 alpha-amylase 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.

4 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 2 3 4 5 6

48 hGA3 soaking

treatmentat 4°C (mM)

0 4º C 0 2 5 7 8 8

500 4ºC 0 7 10 15 19 22

1000 4ºC 0 5 9 13 17 19

1500 4º C 0 6 8 11 16 18

2 weeks soaking treatment at different

temperature

-20°C 4º C 0 0 0 0 0 0

8°C 8ºC 0 0 1 2 2 3

24ºC 24ºC 0 0 0 1 1 1

Gibberellic acid and cold effect of seed of Ferula gummosa 5

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

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 GA3 after two (1), four (2) and six weeks (3) of culture at 4°C; seeds pretreated with 1000mM GA3 after two (4), four(5) and six weeks (6) of culture at 4°C; seeds pretreated at 4°C after two (7), four (8) and six weeks (9) of culture at 4°C; seeds pretreated at -20°C after six weeks of culture at 4°C (10); seeds pretreated at 8°C after six weeks of culture (11); seeds pretreated at 24°C after two (12), four (13) and six weeks (14) of culture at 24°C. (arrows show corresponding bands with 28 and 55 kDa α-amylase marker peptides).

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

REFERENCESBenson, 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: 758–759.

Ghahreman, A (1986) Flora of Iran, Tehran: RIFR Publication.

Hilhorst, H.W.M (1993). New aspects of seed dormancy. In Fourth lnternational Workshop on Seeds, Basic and Applied Aspects of Seed Biology, Côme, D., and Corbineau, F., eds, Paris: ASFIS.

Jacobsen, J.V., and Higgins, T.J.V (1982) 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 Tavakkol-Afshari 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 – 2786.

Zargari, A (1991) Medicinal Plants Tehran: Tehran University Publication.

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

ABSTRACTIn this study we examined effects of the different concentrations of 17-β estradiol and progesterone (0 (control), 1.5, 3.02, 6.05 μM) 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 μM progesterone treatment, the colony’s diameters were higher than the other treatments. In 1.5 μM estradiol, fruit body formation was stimulated on the 12th day after treatment. In this treatment (1.5 μM 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 .

INTRODUCTIONPleurotus 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 is a 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.irTel : +9821-44865323received: July, 2010Accepted: 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 of lignin and phenol 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 the effects of 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.

In this study effects of the different concentrations of 17-β estradiol and progesterone (0 (control), 1.5, 3.02, 6.05 μM) were examined on the growth rate and fruit body formation of Pleurotus florida .

MATERIALS AND METHODSFungal 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 27°C and 70% relative humidity. After the colonies reach a diameter of 25 to 35 mm, the plates are inverted and incubated at 12 to 15°C .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 µl of hormone added into well. Triplicates of each plate have been performed. The plates were incubated at 37°C 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 Duncan’s multiple range tests (Snedecor and Cochran , 1987).

RESULTSThe 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 µM progesterone in the 6th day after inoculation and 3.02 µM 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 µM esteradiol and 6.05 µM 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 µM estradiol. Generally, it was observed that the highest concentration progesterone (6.05 µM) increased the protein content of P. florida higher than the other treatments and its not significantly different from protein content of 1.5 µm estradiol and 3.02 µM progesterone treatments.The results of protein profile by SDS-Page showed that in 1.5µM estradiol, 1.5 and 3.02 µM, progesterone was a 28 KD protein band which is not present in control and the other treatments (Fig 5). In 1.5 µM progesterone treatment showed the most clear polypeptide band.

DICUSSIONBased on the results of this study, it can be concluded P.florida has esteroidal receptors and 17 β-estradiol and progesterone treatments has effects of fruit body formation. The identification of the genes and proteins involved in fruiting, as well as studying the effects of environmental and biochemical treatments on fruit body formation are extremely important biotechnologically and commercially. Hormones control and coordinate complex 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 hormone-bound 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 17b-HSD (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

The effect of steroid hormone on Pleurotus florida 9

and quinones is only suspected (Iwata, 1989). In this study we showed protein content increased in

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 µM progesterone. c 3.02 µM progesterone. d 6.05 µM progesterone. e 1.5 µM estradiol. f 3.02 µM estradiol. g 6.05 µM estradiol.

H o rm o n a l tre a tm e n ts (µM )

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

C o n tro l pro g e s tro n e1 .5

pro g e s tro n e3 .0 2

pro g e s tro n e6 .0 5

e s tra di o l 1 .5 e s tra di o l 3 .0 2 e s tra di o l 6 .0 5

Dia

met

er o

f co

loni

es (

cm)

6 da y s7 da y s

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 µM estradiol. c 6.05 µM estradiol. d 3.02 µM progesterone.

0

2

4

6

8

10

12

14

control 1.5 est 3.02 est 6.05 est 1.5 pro 3.02 pro 6.05 pro

hormonal treatments(microM)

prot

ein

cont

ent(m

M)

The effect of steroid hormone on Pleurotus florida 11

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

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

REFERENCESBezalel, L. ,Hadar, Y|., Cerniglia, CE. (1996)

Mineralization of Polycyclic Aromatic Hydrocarbons by the White Rot Fungus Pleurotus ostreatus , Appli.Environ. Microbiol. 62: 292-295.

Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilization the principle of protein-dye binding.Annal.Biochem 72:248-254.

Chory, Li. J. (1999) Brassinosteroid actions in plants. J. Exp. Bot . 50:275–282.

Fountoulakis, MS., Dokianakis, SN., Kornaros, ME., Aggelis, GG., Lyberatos, G. (2002) Removal of phenolics in olive mill wastewaters using the white-rot fungus Pleurotus ostreatus. Water Res. 36: 4735-4744 .

Iwata N., Inazu N., Satoh T. (1989) The purification and properties of NADPH-dependent carbonyl reductases from rat ovary. J Biochem. 105:556-564

Jonathan, S.G. and Fasidi, I.O. (2003) Requirements for vegetative Growth of Tricholoma lobayensis (Heim), A Nigerian Edible Fungus. Adv. Food Sci. 25: 91- 95.

Lanisnik, T., Zakelj-Mavric, M., Belic, I. (1992) Fungal 17ß-hydroxysteroid dehydrogenase. FEMS Microbiol. Lett.. 99: 49-52.

Mckenna, NJ, O'Malley, BW. (2002) Combinatorial control of gene expression by nuclear eceptors and coregulators.Cell. 108:465–474.

Nayana, Jose , Janardhanan, KK.(2000) Antioxidant and antitumour activity of Pleurotus florida. Current Science. 79(7): 941-943.

Pare, PW., Tomlinson, JH.(1999) Plant volatiles as a defense against insect herbivores. Plant Physiol. 121:325–331.

Snedecor, G.W., Cochran, G.W. (1987) Statistical Methods .Oxford IBH Publishing Co. Ltd, New Delhi : 20-25.

Solomko, EF., Eliseeva. GS. (1988) Biosynthesis of vitamins B by the fungus Pleurotus ostreatus in a submerged culture. Prikl Biokhim Mikrobiol. 24: 164-169.

Wolter, M., Zadrazil, R., Martens and Bahadir, M.(1997) Degradates of eight highly condensed polycyclic aromatic hydrocarbon by Pleurotus florida in solid 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, Iran2-Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science

University of Malaya ,50603 Kuala Lumpur ,Malaysia

ABSTRACTSoil 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 µM 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.

INTRODUCTIONSalinity 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.irTel : +98-912-3803218Received: July, 2010

Accepted: September, 2010cope 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 water potential through several cellular compartments (Greenway and Munns ,1980; Marschner , 1990 ; Robinson et al, 1997; Serraj and Sinclair ,2002).In addition to their role in cell water relations , organic solutes accumulation and also contribute to the maintains of ionic homeostasis and stabilization of some macromolecules and organelles such as

proteins , protein complexes 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 METHODSPlant 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 µM] immediately. The initial pH of the medium was adjusted to 5.8 before autoclaving. The cultures were incubated at 25±3°C with 16h light/ 8h dark with a light intensity of 400 µmoles 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 70°C 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 determinationChlorophylls were extracted with 80% acetone under

a dim green light and determined 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= ΔM×Δt

Where Δ 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 Δt period (Hunt, 1990) according to the equation 3.

Response of Musa acuminata var. Mas to interaction of salinity and cadmium 15

Equation 3: M=ΔM/ΔLn (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 analysisAnalysis of variance and mean comparison

procedures was used to detect differences between treatments. Mean separation procedures were carried out using the ANOVA test with SPSS (ver.14) significant difference at (P< 0.05).

RSULTSTable 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 µM 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+(µM)Salinity

(mM)

RFW

(g/Plant)

RDW

(g/Plant)

SFW

(g/Plant)

SDW

(g/Plant)

WC

(ml/g)DW

0 0

5075100125150

0.613±0.87g

0.348±0.004cde

0.285±0.009bcd

0.224±0.01ab

0.368±0.019def

0.469±0.035f

0.0065±0.003f

0.0005±0.0003abc

0.0015±0.0008abc

0.003±0.0017a

0.005±0.0028de

0.021±0.012f

1.039±0.03g

0.4±0.04ef

0.364±0.016cdef

0.238±0.03bcdef

0.248±0.006ab

0.359±0.012cdef

0.114±0.0005h

0.038±0.006g

0.032±0.018fg

0.0252±0.0017cdef

0.02±0.0003bcde

0.0316±0.0012fg

10.32±2.01bcdeg

9.28±0.79abcdefg

8.69±0.59abcdef

8.05±0.95abcd

7.50±1.45abc

7.33±1.00abc

25 0

5075100125150

0.309±0.047bcd

0.613±0.033def

0.251±0.016abc

0.23±0.025ab

0.227±0.008ab

0.317±0.055bcd

0.011±0.006cd

0.0061±0.0035abc

0.0036±0.002ab

0.002±0.0012a

0.008±0.0047abc

0.0094±0.0054ab

0.232±0.009ab

0.201±0.09a

0.324±0.011bcdef

0.43±0.06f

0.233±0.003ab

0.224±0.015ab

0.0262±0.0027def

0.037±0.0015g

0.0206±0.0016bcde

0.031±0.0018fg

0.0126±0.0003a

0.0126±0.0006a

6.52±0.59a

7.14±2.54ab

11.67±0.73defg

10.99±1.1cdefg

8.68±1.24abcdef

12.37±5.96fg

50 0

5075100125150

0.162±0.016a

0.359±0.034cde

0.278±0.015bcd

0.336±0.033bcd

0.451±0.035ef

0.226±0.004ab

0.008±0.0046bc

0.0049±0.0028ab

0.003±0.001abc

0.0023±0.001abc

0.0083±0.004ef

0.0032±0.0018ab

0.209±0.0085abcde

0.268±0.011abcd

0.256±0.025abc

0.366±0.029def

0.371±0.014def

0.290±0.030abcd

0.0193±0.0006abcd

0.0166±0.0006ab

0.019±0.0015abc

0.022±0.001bcde

0.027±0.0023ef

0.024±0.0023cde

6.98±1.55ab

12.51±0.25g

9.65±1.25abcdefg

12.03±2.5efg

8.43±0.87abcde

8.44±0.81abcde

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

Ion content (ppm/gDW)

NaCl Cadmium content(µM)(mM) 0 25 50

Chl a (mg/g FW) 05075100125150

0.61±0.02e

0.43±0.01cd

0.13±0.004a

0.48±0.2cde

0.33±02bc

0.54±.006de

0.35±0.03bcd

0.37±0.01bcd

0.66±0.01e

0.49±0.02cde

0.37±0.01bcd

0.35±0.003bc

0.37±0.01bcd

0.24±0.007ab

1.68±0.03g

4.08±0.08h

1.44±0.04f

1.41±0.01f

Chl b(mg/g FW) 05075100125150

0.16±0.02a

0.16±0.003a

0.21±0.07a

0.36±0.05a

0.12±0.004a

0.32±0.04a

0.18±0.02a

0.18±0006a

0.30±0.02a

0.10±0.009a

0.11±0.004a

0.22±0.03a

0.13±0.006a

0.13±0.008a

0.76±0.35b

0.16±0.04c

0.87±0.01b

0.90±0.01b

Proline content(mg/g leaf FW)

05075100125150

66.93±0.75a

87.46±0.40bc

130.77±1.2h

152.11±1.96i

175.6±1.87j

194.75±1.79k

88.20±3.18bc

97.94±2.26e

118.02±1.88g

189.70±3.66k

193.00±2.84k

203.24±3.23l

94.88±2.67cde

90.50±2.00cd

81.99±2.10b

107.31±0.63f

89.58±4.13cd

96.11±1.91de

RGR (g/gday) 05075100125150

0.033±0.001i

-0.0115±0.0001h

-0.0212±0.0007g

-0.0653±0.001a

-0.0427±0.001e

-0.0184±0.0002g

-0.0337±0.0008f

-0.0393±0.002e

-0.0497±0.0008d

-0.0317±0.002f

-0.0663±0.0008a

-0.0697±0.0008a

-0.0570±0.001b

-0.0680±0.001a

-0.0543±0.002bc

-0.0523±0.001cd

-0.0307±0.001f

-0.0570±0.001b

ULR(g /m2day) 050

0.04±0.002j

-0.009±0.0005i

-0.03±0.001f

-0.03±0.0008f

-0.04±0.001de

-0.06±0.001a

Response of Musa acuminata var. Mas to interaction of salinity and cadmium 17

75100125150

-0.02±0.001gh

-0.04±0.001cde

-0.04±0.001e

-0.02±0.0005h

-0.04±0.0005e

-0.02±0.001fg

-0.06±0.005a

-0.04±0.0005e

-0.05±0.002bc

-0.05±0.001bcd

-0.03±0.001fg

-0.05±0.0008b

SI 05075100125150

-------59.92±3.65cd

-63.38±0.54bc

-73.04±1.55a

-59.38±1.58cd

-45.35±7.16e

-60.47±3.65cd

-60.47±1.27cd

-75.04±2.02a

-69.94±1.66ab

-73.58±2.64a

-76.32±3.11a

-67.94±2.21abc

-74.68±1.73a

-72.49±1.01a

-70.30±1.31ab

-52.09±3.17de

-70.12±0.36ab

Leaf Area(cm2/plant)

05075100125150

7.91±0.5abcd

9.52±0.41cdef

9.71±0.22def

8.26±0.88abcde

8.36±0.29abcdef

9.28±0.25bcdef

7.35±0.49abcd

11.16±0.08fg

12.60±1.65g

13.15±1.36g

7.49±0.67abcd

6.66±0.08abc

10.92±0.98efg

7.44±1.24abcd

6.08±0.79a

7.97±0.52abcd

6.37±0.14ab

7.34±1.84abcd

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 Cadmium content(µM)(mM) 0 25 50

Cd content in roots 05075100125150

0.00a

0.00a

0.00a

0.00a

0.00a

0.00a

17.181±0.219i

44.167±0.167m

15.605 ±0.049g

13.25 ±0.055f

5.716 ±0.045b

6.813 ±0.008c

21.227 ± .107j

22.667 ± 0.083k

26.41± 0.164l

16.894±0.161h

10.486 ±0.075d

10.909±0.041e

Cd content in shoots 05075100125150

0.00a

0.00a

0.00a

0.00a

0.00a

0.00a

2.635±0.015d

3.778±.0277e

0.00a

0.00a

0.00a

1.490±0.019b

28.333±0.119l

10.151±0.03i

7.209±0.024h

5.291±0.024g

2.009±0.018c

4.952±0.047f

Ca content in roots 05075100125150

314.40 ±4.17k

344.49±1.15l

138.08±3.03a

360.00±9.51m

281.42±4.52j

141.33±1.25ab

190.76±0.57f

485.66±2.02n

219.87±0.89g

197.97±1.65f

190.12±0.53f

145.20±0.9abc

148.46±1.44bc

152.36±0.4cd

232.72±0.9h

246.36±0.69i

159.33±1.07d

171.03±3.1e

Ca content in shoots 05075100125150

696.11±5.61i

330.98±1.67b

424.53±14.18f

336.88±3.05bc

602.66±14.81g

370.0±2.52de

350.47±0.82bcd

650.83±15.46h

373.11±1.45e

806.25±5.72j

897.38±9.19k

331.17±1.35b

358.45±1.25cde

377.42±2.1e

344.56±0.49bc

347.63±4.52bc

267.65±2.77a

1315.71±1.42l

Na content in roots 05075100125150

166.66±0.83b

180.47±1.03c

340.43±1.09f

352.00±0.65g

564.60±2.55m

590.66±1.01n

93.61±0.68a

282.43±0.48d

359.62±1.49h

402.00±3.21j

428.88±0.77k

369.40±1.37i

96.46±0.69a

180.35±2.28c

332.19±0.41e353.79±1.89g

435.30±0.75l

436.66±1.18l

Na content in shoots 05075100125150

172.64±0.58b

247.22±0.32d

282.00±1.17f

320.66±0.13h

600.88±2.11m

865.66±4.84p

229.36±0.15c

457.55±0.32k

545.00±3.36l

738.23±1.17n

773.09±0.13o

982.50±0.58q

180.23±4.84b

266.66±7.42e

304.81±0.15g

357.57±2.11i

391.25±3.36j

609.95±3.81a

k content in shoots 05075100125150

180.89±0.61k

113.03±0.54i

109.85±0.32h

80.74±0.24e

76.11±0.55d

52.48±0.11b

181.12±0.64k

115.57±0.22ij

96.18±0.44fg

76.3±0.13d

71.57±0.23c

45.36±0.08

213.14±1.5h

95.66±0.18f

114.59±0.43ij

116.16±0.46j

98.67±0.18g

52.20±3.1b

k content in roots 05075100125150

49.76±0.39m

36.22±0.43h

32.53±0.19f

28.60±0.21d

25.09±0.04c

15.11±0.06a

45.11±0.06l

42.20±0.02k

39.11±0.07i

31.45±0.19e

32.31±0.12f

22.45±0.27b

55.52±0.25n

58.51±0.12o

45.38±0.23l

40.34±0.15j

34.36±0.14g

28.30±0.12d

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

DISCUSIONSalinity 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 over-expression 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 44.5µM 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(Prasad and Haegemeyer,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 Cd- treated 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.

ACKNOWLEDGMENTSThis 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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218:1-14

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.

ABSTRACTThis 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.

INTRODUCTIONLichens 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 don’t 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.irTel : +98-21-44364066Received: July, 2010Accepted: 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 non-exposed 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.

MATERLALS AND METHODSSample collection

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 36°12'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.

Producing electromagnetic FieldsBecause of the size of lichens, a plastic spool at 4×4cm 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 frequency, 150v and 0.24A for 4 hours during a 2 or 3 days period. Lichens grow naturally in the earth’s magnetic field which its intensity is 0.5G. The device used in this experiment was able to produce stronger magnetic fields than earth’s 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:

= The permeability factor in vacuum which is equal to

= The nuclear permeability factor in 100v is about 13128 and in 150v is about 12491.

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).

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

Pigment assays

Total chlorophyll content was determined using Arnon’s 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. 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. FW.

Proline assay

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

Parietin assayParietin was determined by Solhaug &Gauslaa (2001) methods. The absorbance was measured spectrophotometrically at 434nm. Parietin was calculated base on

Electromagnetic effect on Lichen species 25

Statistical analysis

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.

RESULTSTotal 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

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. .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 bbase on mg. .FW is significant at p <0.05 in both speciesexcept the increase of 15mT stressed sample in L. lobificans.

Fig. III. Effect of electromagnetic fields on the carotene base on mg. .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 onµMol. .FW. is significant at p <0.05 and the lowestamount was shown in stressed samples in L. lobificans.

Fig. VI. Effect of electromagnetic fields on the base on parietin is significant at p <0.05.

DISCUSSIONThe 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 didn’t 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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30

Study of Eucalyptus Allelopathy Effect on Morphophysiological Parameters of Brassica napus L.

Maryam Niakan* and Kolsum SaberiDepartment 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 .

INTRODUCTIONThe 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.comReceived: May, 2010

Accepted: August, 2010Eucalyptus is one of the most important tree species for wood production in world. It is said that Eucalyptus is toxic (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 PreparationFive 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 PlantedCanola 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 ºC 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 ºC 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 25°C 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 p≤0.01 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 p≤0.01 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 p≤0.01 (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) .α-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). α-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 α-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 response depends on the allelochemicals 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, a p-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). ρ-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 , 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 Shoot

Control

Decompose (3%)

Decompose (6%)

Length F.W D.W 7.8a 1.493a 0.293a

5.567b 0.809b 0.121b

4.917b 0.271c 0.045c

Length F.W D.W

3.333b 4.877ab 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 Shoot

Control

aqueous extracts (5%)

aqueous extracts (15%)

aqueous extracts (30%)

Length F.W D.W 9.875 ab 2.138a 0.339a

8.838 b 1.695a 0.3a

9.143 b 1.376a 0.308a

10.975 a 1.609a 0.32a

Length F.W D.W

6.667c 4.379a 0.709ab

5.788c 3.648a 0.649b

9.238b 5.109a 0.773ab

11.825a 5.906a 1.049a

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 15 30

Eucalyptus extract concentration(%)

chl c

onte

nt

(mg

.g-1

FW)

Chl a

Chlb

a

bbab

ab

a

ab

0

0.1

0.2

0.3

0.4

0.5

0.6

شاهد 3 6

Eucalyptus leaf decompose (%)

chl c

onte

nt

(mg

.g-1

FW)

Chl a

Chlbb

a

a

b

ab

a

(A) (B)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 5 15 30

Eucalyptus leaf extract (%)

chl c

onte

nt

(mg

.g-1

FW)

Chla+ Chlb

a/b

d

ba

c

a

c bc b

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

شاهد 3 6

Eucalyptus leaf decompose concentration(%)

chl c

onte

nt

(mg

.g-1

FW)

Chla+ Chlb

a/b

bb

a

a a a

(C) (D)

Fig Ι): 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

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 15 30

Eucalyptus leaf extract (%)

solu

ble

suga

r (m

g.g

-1DW

)

leaf

rootb

a

ababa

a

aa

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

شاهد 3 6

Eucalyptus leaf decompose(%)

solu

ble su

gar

(mg

.g-1

DW

)

leaf

root

a

a

bb

ab

ab

(F) (E)

0

1

2

3

4

5

6

7

0 5 15 30

Eucalyptus leaf extract(%)

Phen

olic

com

poun

ds c

onte

nt

( m

g.g

-1.F

W

)

leafroota

a

aa

b

cbc

a

0

1

2

3

4

5

6

7

8

شاهد 3 6

Eucalyptus leaf decompose(%)

Phen

olic

com

poun

ds c

onte

nt(

mg

.g-1

.FW

)

leaf

roota

aa

aa

a

(G) (H)

Fig Π): 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.

INTRODUCTIONForest 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, 2010Accepted: 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 has been used widely 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 situation will 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 hazel- nut 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 METHODESStudy 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

nutrient uptake and abscission relations in Ash 39

sulfur) are also used in large amounts but are typically readily available in adequate. 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)

12345678910

17

20

18

12

15

15

13

25

23

27

30454025354555505560

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

June July August September

%Dry weight (2002)

%Dry weight (2003)

%Inorganic matter (2002)

%Inorganic matter (2003)

-

31.2

-

8.2

35.2

32.7

10

8.26

44

35.9

14.5

10.74

46.1

41.6

12.5

10.08

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

P Ca K

July

August

September

0.354

0.253

0.049

1.09

2.29

2.35

.946

1.169

1.174

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

P Ca K Mg Na

June

July

August

September

2.65

0.724

0.684

0.506

1.489

1.63

2.09

1.65

0.725

0.601

0.72

0.71

0.235

0.227

0.226

0.222

0.06

0.065

0.071

0.072

Table5- Some chemical properties of soil

K Na Mg Lime% Ca Cl %OC %N %Chalk P

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

REFERENCESBakedjeva, 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 physico- chemical 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: 1-10.

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: 94-103.

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 contentShoarian , 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.

INTRODUCTIONNitrogen is a major element for all living organisms. The use of 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.comReceived: June, 2010Accepted: 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 METHODThe 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.

RESULTThe 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

Root dry weightLeaf dry weight[NO3-]±RhizobiumTREATMENT

0.25a ±

0.27a ± 0.03

0.27a ± 0.02

0.26a ± 0.01

0.25a ± 0.00

0.14b ± 0.01

0.11a ± 0.02

0.11a ± 0.01

0.13a ± 0.03

0.14a ± 0.04

0.15a ± 0.03

0.07b ± 0.01

0.46a ± 0.02

0.46a ± 0.06

0.47a ± 0.02

0.5a ± 0.06

0.49a ± 0.02

0.29b ± 0.02

1mM - Rh.

Rh.+ 1mM

.Rh+2.5mM

Rh. + 5mM

Rh. +10mM

Rh.+ 15mM

Control 1

Control 2

Treatment 1

Treatment 2

Treatment 3

Treatment 4

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 diameternodules of Phaseolus vulgaris inoculated and non-inoculated with rhizobium

Nodule numberNodule diameter[NO3-]±RhizobiumTreatment

0.00d ± 0.00

23.00a ± 3.00

18.33b ± 4.04

6.67c ± 2.89

3.33cd ± 1.15

0.00d ± 0.00

0.00d ± 0.00

2.49a ± 0.3

1.39b ± 0.21

1.02c ± 0.15

0.73c ± 0.14

0.00d ± 0.00

1mM - Rh.

Rh.+ 1mM

.Rh+2.5mM

Rh. + 5mM

Rh. +10mM

Rh.+ 15mM

Control 1

Control 2

Treatment 1

Treatment 2

Treatment 3

Treatment 4

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

LeafStemRoot[NO3-]±RhizobiumTreatment

17.07b ± 1.29

17.85b ± 2.89

17.92b ± 2.46

19.73ab ± 0.01

21.45a ± 1.09

23.03a ± 0.54

16.58a ± 2.26

18.87a ± 1.83

19.85a ± 1.15

18.96a ± 4.38

20.59a ± 2.07

23.33a ± 2.97

22.03a ± 1.95

22.42a ± 2.65

24.04b ± 3.51

25.22b ± 1.34

25.05b ± 2.42

33.17c ± 1.61

1mM - Rh.

Rh.+ 1mM

.Rh +2.5mM

Rh. + 5mM

Rh. + 10mM

Rh.+ 15mM

Control 1

Control 2

Treatment 1

Treatment 2

Treatment 3

Treatment 4

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

LeafStemRoot[NO3-]±RhizobiumTreatment

9.93b ± 0.55

10.99b ± 0.8

13.38b ± 1.13

14.72ab ± 2.62

16.1a ± 4.3

18.33a ± 4.91

16.68b ± 1.11

17.04b ± 3.48

18.45b ± 5.57

20.47b ± 4.48

22.27b ± 6.2

26.26a ± 4.48

24.22b ± 3.43

26.08b ± 4.93

36.64ab ± 1.82

42.6ab ± 3.67

44.39a ± 7.3

50.6a ± 6.92

1mM - Rh.

Rh.+ 1mM

.Rh +2.5mM

Rh. + 5mM

Rh. + 10mM

Rh.+ 15mM

Control 1

Control 2

Treatment 1

Treatment 2

Treatment 3

Treatment 4

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

LeafStemRoot[NO3-]±RhizobiumTreatment

0.36a ± 0.050.46a ± 0.070.47a ± 0.060.89a ± 0.050.98a ± 0.361.29a 0.42

2.1b ± 0.442.38b ± 0.222.5b ± 0.47

2.61b ± 0.532.96ab ± 0.9

3.38a ± 0.378

6.46b ± 1.147.1b ± 1.138.98a ± 1.29.91a ± 0.5910.1a ± 1.111.3a ± 1.12

1mM - Rh.Rh.+ 1mM

2.5mM+Rh.Rh. + 5mMRh. +10mMRh.+ 15mM

Control 1Control 2

Treatment 1Treatment 2Treatment 3Treatment 4

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

LeafStemRoot[NO3-]±RhizobiumTreatment

23.32b ± 2.38

24.39b ± 2.7

25.58b ± 2.26

26.27ab ± 1.77

34.49ab ± 3.14

35.04a± 2.61

27.3b ± 2.37

27.6b ± 2.87

31.34a ± 2.03

31.06a ± 2.74

31.38a ± 1.76

32.2a ± 2.85

10.69 b ± 1.65

13.4b ± 2.88

20.31a ± 1.15

21.1a ± 2.95

22.23a ± 1.87

22.97a ± 1.23

1mM - Rh.

Rh.+ 1mM

.Rh+2.5mM

Rh. + 5mM

Rh. + 10mM

Rh.+ 15mM

Control 1

Control 2

Treatment 1

Treatment 2

Treatment 3

Treatment 4

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 NO3 – on the averages of phosphorus content in root,

stem and leaf of Phaseolus vulgaris inoculated and non-inoculated with rhizobium

LeafStemRoot[NO3-]±RhizobiumTreatment

0.27b ± 0.05

0.45b ± 0.13

0.69a ± 0.11

0.74a ± 0.12

0.88a ± 0.1

0.93a± 0.21

0.6b ± 0.28

0.63b ± 0.3

0.78b ± 0.05

0.91a ± 0.14

0.96a ± 0.07

1.15a ± 0.07

1.02d ± 0.12

1.23d ± 0.48

2.74cd ± 0.47

4.15c ± 0.76

6.48b ± 0.63

8.96a ± 1.30

1mM - Rh.

Rh.+ 1mM

2.5mM + Rh.

Rh. + 5mM

Rh. + 10mM

Rh.+ 15mM

Control 1

Control 2

Treatment 1

Treatment 2

Treatment 3

Treatment 4

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

DISCUSSIONThe 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 47

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).

REFERENESAndrews, M., Faria, S.M. ,Mcinroy, S.G., and

Sprent, J (1990) Constitutive nitrate reductase activity in the leguminosae. Phytochemistry 29:49-54.

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 of legumes. Physiol. Plant,70: 757–765.

Brockwell, J, Bottomley, P. J., Thies, J.E(1995) Manipulation of rhizobia microflora for improving legume productivity and soil fertility: a critical assessment. Plant Soil ,174:143–180.

Carolus, Robert L(1938) Effect of certain ions, used singly and in combination, on the growth

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 KhanFood and Biotchnology research Centre, Pakistan Council of Scientific and Industrial

Research Laboratories Complex, Lahore, Pakistan

ABSTRACTThe 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 plantMuafia 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.comReceived: July, 2010Accepted: 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 METHODSSweet 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 6- Benzyl amino-purine (BA) in combination with 1.07μM Nephthalene acetic acid (NAA). BA was supplied from 0.44 μM to 8.88μM. After inoculation these cultures were kept for 21 days in a growth chamber maintained at 25±2°C under 16h photoperiod and 48μmol 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 basilicum L.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 4°C. For antibacterial assay 24h old bacterial cultures at 37±1 ºC 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 30μL of each plant extract were placed on pre-inoculated culture media and incubated at 37±1ºC for 24h. All experiments were performed in duplicate. Penicillin, co-trimoxazole and Streptomycin were used as positive controls. 25μL of each antibiotic solution (0.01g/10mL) was dropped on paper discs. 25μL of 10% aqueous solution of dimethylsulfoxide (DMSO) was used as negative control during this study.

RESULTS AND DISCUSSIONEffect of different concentrations of BA (0.44-8.88µM) in combination with NAA (1.07 µM) 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 (8.88µM) along

with NAA (1.07 µM) in terms of callus induction (100% explants). Whereas lesser quantity of BA (0.88µM) in the presence of 1.07 µM 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.

Sr.#Medium

composition

(μM)

Callus initiation

%

Callus growth

Morphogenetic potential Remarks

1MS+1.07NAA

+0.44BA40 + + + Root initiation Callus Compact green

2MS+1.07NAA

+0.88BA

38 + Nil Callus Light green &loose

3MS+1.07NAA

+2.22BA

70 +++ Nil Callus some what yellowish vitrifaction

4MS+1.07NAA

+4.44 BA

50 ++ Root initiation Callus Compact whitish green

5MS+1.07NAA

+8.88 BA

100 ++++ NilCallus Compact granular light green with powdery

upper surface

+ = Poor

+ + = Good

+ + + = Very Good

+ + + + = Excellent

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

Micro-organisms Zone of inhibition (mm)

MCE ACE MLE ALE DMSStreptomycin

25µg/disc

Penicillin

25µg/disc

Cotrimoxazole

25µg/disc

Escherichia coli ATCC 25922 - - - - - 21 - -

Bacillus subtilis ATCC 6633 12.5 11.5 9.0 8.5 - 28 16.5 -

Klebsiella pneumoniae HI - - - - - 18.5 - -

Salmonella paratyphi HI - - - - - 14 - -

Pseudomonas aeruginosa HI - - - - - 22 - -

Staphylococcus aureus HI 8.0 7.5 - - - 19 26 -

Escherichia coli HI - - - - - 24 - -

Enterobacter species HI - - - - - 22 - -

Absolute Methanol callus Extract = MCE70% aqueous Methanol callus extract = ACE Absolute Methanol Leaves extract = MLE70% aqueous Methanol Leaves extract = ALEHospital 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, 493-496.

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, 473–497.

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 –80°C for later processing. Ground tissue powder can also be stored at –80°C. If your lysate is too viscous, reduce the amount of starting material or increase the amount of extraction buffer like CTAB buffer.

AbbreviationTE buffer: Tris-Cl. EDTAFISH: Fluorescence In Situ HybridizationRFLP: Restriction Fragment Length PolymorphismEtBr: Ethidium BromideCTAB: CylTrimethylAmmonium BromidePCR: Polymerase Chain Reaction

Read more onSambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press Sambrook and Russellhttp://serc.carleton.edu

http://www.accessexcellence.orghttp://www.protocol-online.org

http://www.ambion.com/techlib

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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

IJPPIranian Journal of Plant Physiology

Managing Editor: Mozhgan Farzami Sepehr(PhD)

Assistant ProfessorDepartment of BiologyFaculty of AgricultureIslamic Azad University Saveh BranchSaveh, Iranfarzamisepehr@iau-saveh.ac.ir

Editor in Chief: Mahlagha Ghorbanli(PhD)

ProfessorDepartment of BiologyFaculty of ScienceIslamic Azad University Gorgan BranchGorgan, Iranghorbanli@yahoo.com

Executive Editor:

Mona Farhadi(PhD)

Assistant ProfessorDepartment of BiologyFaculty of AgricultureIslamic Azad University Saveh BranchSaveh, Iranmonafarhadi@yahoo.com

Editorial Board:Iftikhar Hussain Khalil (PhD)Professor

Plant Breeding and Genetics DepartmentNWFP Agricultural University

Peshawar, Pakistan

(www.aup.edu.pk)drihkhalil@gmail.comJennifer Ann Harikrishna (PhD)

Associate Professor, Genetics and Molecular BiologyInstitute of Biological SciencesFaculty of ScienceUniversity of Malaya50603 Kuala LumpurMalaysiajennihari@um.edu.my

Gholamreza Bakhshi Khaniki (PhD)

ProfessorFaculty of AgriculturePayame Noor UniversityLashkarak RoadTehran , IranP.O.Box:14335-333  Fariba Meighani(PhD)

Assistant ProfessorIranian Research Institute of Plant Protectionfmaighany@yahoo.com Maryam Shahbazi (PhD)

Assistant ProfessorMolecular physiology Department,Agriculture Biotechnology Research Institute of Iran (ABRII)Mahdasht RoadP. O. Box 31535-1897Karaj, Iran

MShahbazi@abrii.ac.ir

http://www.abrii.ac.ir

BOOK REVTEW 57

Seyed Mohammad Mahdi Hamdi (PhD)

Assistant ProfessorDep. of BiologyFaculty of ScienceIslamic Azad University of GarmsarGarmsar, Iranmm_hamdi@asia.com

Mohamad Ali Baghestani Meibodi(PhD)

Associate ProfessorIranian 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.

Aims and scopeThis 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 analyzing experimental results. Papers outlining trends and hypotheses are accepted as well. Brief communications are not accepted. However, in some cases, the editors may suggest that authors shorten a manuscript to the size of a brief communication (no more than 10 pages of text and 4 figures and/or tables in all). Manuscript submission implies that the material

has not been published before and is not under consideration for publication anywhere else.

Manuscript requirementsManuscript length should not exceed 10 printed pages (reviews not more than 20 pages), including references, tables, and figure captions; it should contain no more than 7 figures. The manuscript must be typed (Times New Roman font, 12 pt, 1.5 spacing throughout) in a single column on one side of white paper (A4, 210 × 297 mm) with left and top margins of 2.5 cm and a right margin of 1.5 cm. All pages, including references, tables, and figure captions, should be numbered consecutively in the top right-hand corner. All lines should be enumerated throughout the entire text.Please arrange your manuscript as follows: title, author(s), affiliation(s), abstract, INTRODUCTION, MATERIALS AND METHODS, RESULTS, DISCUSSION, ACKNOWLEDGMENTS, REFERENCES, TABLES, FIGURE CAPTIONS.The title must be concise (no more than 10 words) but informative. Capitalize the first letters in all nouns, pronouns, adjectives, verbs, adverbs, and subordinate conjunctions. Avoid nonstandard abbreviations.Authors’ initials and surnames should be written with one space between the initials and between the initials and an author’s surname and with the conjunction “and” before the last author. Author affiliations should be marked as 1,2 etc. On a separate page, provide the full names of all authors, their postal addresses and telephone and fax numbers, as well as e-mail addresses, and indicate the corresponding author.Author affiliations include the department, institution, and complete address of each author. The fax number and e-mail address of the corresponding author should be indicated after his or her postal address.

AbstractAll papers, including Brief communications, should be preceded by a concise (of no more than 250 words) but informative abstract, in which the plant material (binomial, including authority) is given. The abstract should explain to the general

reader the major contributions of the article. The abstract is typed as a single paragraph. Citing and discussing literature arenot recommended.

Key words(No more than 7 items) are listed beginning with the Latin name(s) of the organism(s) studied

without author’s name and arranged as follows: Lycopersicon esculentum - transgenic tomato plant - ethylene

Abbreviationsare listed in alphabetical order and arranged as follows: BA—benzyladenine; PSI—photosystem I; WT—wild type. Define nonstandard abbreviations when they are first mentioned in the text and abstract.

Main headingsWithin the text (INTRODUCTION, MATERIALS AND METHODS, etc.) should be placed

on separate lines and written in all capitals. First-level subheadings should follow title capitalization (example: Cytokinin-Dependent Signal Transduction) and be placed on separate lines. Second-level subheadings (i.e., headings run into a paragraph) should follow sentence capitalization (example: Plant material.).

INTRODUCTION: The introductory part of the article should explain its objective and cite relevant articles published previously.

MATERIALS AND METHODS::should include complete botanical names (genus, species, authority for the binomial, and, when appropriate, cultivar) for all plants studied. Following first mentions, generic names should be abbreviated to the initial except when confusion could arise by reference to genera with the same initial. Growth conditions must be described. New procedures should be described in sufficient detail to be

repeated. A short description of other procedures should also be given. Avoid references like “… as described in “Brown et al(2009)” or “… according

to Tomas and Singh(1996)” This section should also contain the names of the manufacturers (including country name) of materials and reagents. Statistical analysis of the results should be described. Identify the number of replications and the number of times individual experiments were duplicated. It should be clearly stated whether the standard deviation or the standard error is used.

RESULTS: should be presented mainly in figures and tables without their detailed discussion. Double documentation of the same points in figures and tables is not acceptable.

DISCUSSION: should contain an interpretation but not a recapitulation of the results. The Results and Discussion sections may be combined if a description of experimental results is brief or when the interpretation of the previous experiment is required for the logical substantiation of the next one.

ACKNOWLEDGMENTS: List dedications, acknowledgments, and funding sources.

REFERENCES: Cite published papers and books; citing the abstracts of meetings is not recommended. References at the end of the paper should be arranged alphabetically (by authors' names) in the reference list, all authors should be named unless there are 10 or more. For titles in English, including titles of books, journals, articles, chapters, and dissertations and names of conferences, use title capitalization. For titles given in a foreign language, follow the rules of capitalization for that language.

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: 9–14.

For correct abbreviations of journal titles, refer to

Chemical Abstracts Service Source Index (CASSI).

Books should be cited as follows:

Cobb, A (1992) Herbicides and Plant Physiology London: Chapman and Hall.

BOOK REVTEW 59

Articles or chapters in books should be cited as follows:

Lichtenthaler, H(1996) Vegetation Stress: An Introduction to the Stress Concept in Plants,

Vegetation Stress, Lichtenthaler, H., Ed., Stuttgart: Gustav Fisher, 4–14.

Dissertations should be cited as follows:

Nesterova, A.N(1989) Effects of Lead, Cadmium, and Zinc Ions on the Meristem Cell Arrangement and Growth of Maize Seedling Roots, Cand. Sci. (Biol.) Dissertation, Moscow: Mosk. Gos. Univ.

TABLES: Each table should have a brief title, be on a separate page, and be 1.5-spaced. Each column should have a heading; units should appear under the column heading(s). Some remarks may be written below the table, but they should not repeat details given in the Materials and Methods section.

FIGURE CAPTIONS: must be a brief self-sufficient explanation of the illustrations. Provide them separately from figures.

FIGURES: All figures (photographs, graphs, and diagrams) should be cited in the text and numbered consecutively throughout. Figures should provide enough information to easily understand them. Figure parts should be identified by lowercase roman letters (I, II, etc.) in parentheses. The axes of each graph should have the numerical scale and the measured quantity with units (for example, CO2 absorbance, μ molm-2s-1), but not photosynthesis, μmol/m-2s-1)). The curves should be defined by italic numbers, and their explanation should be provided in the caption. Submit all figures on separate pages. Supply figures at final size widths: 80 mm (single column) or 160 mm (double column). Maximum depth is 230 mm. Figure number, author’s name, and manuscript title should be written in the bottom left-hand corner.

The manuscript should be signed by all authors.

The electronic version is formed as a complete manuscript file, without figures. Text files should be submitted in Microsoft Word 6.0 or a later version, using Times New Roman font of 12 point size. Submit figures as separate files. The preferred

figure format is TIFF, but JPEG and GIF are also permitted. Load your figures at 600 dpi (dots per inch) for linear

and no less than 300 dpi for halftones and photos. Try to keep files under 5 MB.

Editorial processing (reviewing, editing, and proofs).The Editorial Office informs authors by e-mail that a manuscript is received. Manuscripts prepared incorrectly or in poor English are not considered. All manuscripts submitted will be reviewed. The reviewer evaluates the manuscript, suggests improvements, and recommends accepting or rejecting the paper. Manuscripts and reviewer’s comments are emailed to the authors. Revised manuscripts (two copies and the initial version, along with point-by-point responses to the referee) should be returned within 40 days; otherwise, they will be treated as new submissions. If the revised manuscript is not received within four months, it is rejected. The manuscript is then subjected to scientific editing. Accepted manuscripts are published in correspondence with the date of their receiving. Papers containing new information of exceptional significance may be, on the proposal of the Editor in Chief, published first in the shortest possible time. Manuscripts sent to the Editorial Office are not returned to the authors.The Publishing House will deliver the page proofs to authors electronically only to a single address indicated in the affiliation section.Manuscript submission: Two copies of the manuscript should be mailed to the following address: The Editorial Office of Iranian Journal of Plant physiology, Faculty of Agriculture, Islamic Azad University of Saveh Branch, Saveh , Iran . An electronic version should be sent as an attachment to the following e-mail address:IJPP@iau-saveh.ac.ir

BOOK REVTEW 61

Islamic Azad University Saveh Branch PublisherCopyright Transfer Agreement and ethical requirements for the submitted paper_________________________________________________________________________Copyright

The copyright of this article is transferred to the Islamic Azad University Saveh Branch Publisher effective if and when the article is accepted for publication. The copyright transfer covers the exclusive right to reproduce and distribute the article, including reprints, translations, photographic reproductions, microform, electronic form or any other reproductions of similar nature. The author warrants that this contribution is original and that he/she has full power to make this grant. The corresponding author signs for and accepts responsibility for releasing this material on behalf of any and all co-authors. The authors and their employers retain full rights to reuse their material for their own purposes, with acknowledgement of its original publication in the journal.Ethical requirements for the submitted paper• All research or methodologies identified as being conducted or developed by the authors or institutions will in fact have been so conducted or developed.• Relevant prior and existing research and methodologies will be properly identified and referenced using the standard bibliographic and scientific conventions.• All the content of the submitted paper shall be the original work of the authors and shall not plagiarize the work of others. Short quotes from the work of others should be properly referenced with full bibliographic details of the quoted work. To quote or copy text or illustrations beyond a “short quote” will require the author to obtain permission from the rights holder.• Duplicate submission of the same paper to more than one scholarly journal while the decision from another journal on that same paper is still pending, as well as reporting the same results in somewhat different form, is prohibited.• Authors should take care not to defame other researchers in a personal sense.• Co-authors should be properly and appropriately identified. To be identified as a co-author, the participant 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.

Title of article:Author(s):Author’s signature:Author’s email: Date