Journal of Life Sciences

1. Introduction

Modified Halsey (MHAE), Modified Henderson Rice is the major grain in China, with its annual

(MHE), and Modified Oswin (MOE) for rough and production being around 180 million metric tons in

brown rice from three sources, and found except the recent years. Due to the large population, a portion of

MHAE, the other three equations could fit some or all the rough rice grain is stored for a longer period of time

of the rice data. Sun [2] analyzed 17 source sets of rice (2 years) in China than in developed countries, with

EMC/ERH data with four commonly cited models, i.e., deterioration controlled largely through moisture

MCPE, MHE, MOE, and Strohman-Yoerger (STYE), content and temperature. In order to describe the

and considered the STYE as the preferred equation. In process of drying, the ambient cooling of grain, and

the 2006 version of the ASABE Standard D245.5 “Moisture Relationships of Plant-Based Agricultural

Corresponding author: Xingjun Li, Ph.D., associate professor, research field: cereal grain biochemistry. E-mail:

Products”, the MCPE, MHE, and MOE were lxj@chinagrain.org.

16 The Sorption Isosteric Heats of Rough Rice in China

recommended the EMC/ERH data of rough rice [5].

Table 1 The equilibrium relative humidity (%) produced

However, such suitable equation for Chinese rice by nine saturated salt solutions.

varieties is rather deficient. salt solution a 10 ℃ 15 ℃ 20 ℃ 25 ℃ 30 ℃ 35 ℃ Knowledge of the heat of sorption is important in

understanding the mechanism of grain sorption. It is a

Potassium

23.38 23.40 23.11 22.51 21.61 21.50 Design of more efficient drying systems could be Magnesium

valuable tool in designing the drying process [6].

acetate

33.47 33.3 33.07 32.78 32.44 32.05 achieved by stating the correct mathematical models to

chloride

43.14 43.15 43.16 43.16 43.17 43.16 estimate the heat and mass transfer mechanisms [7-8].

Potassium carbonate

The purpose of the present work is to determine the 57.36 55.87 54.38 52.89 51.40 49.91 most suitable ERH/EMC model corresponding to the Cupric

Magnesium nitrate

chloride

sorption isotherms of rough rice of thirteen varieties in

Sodium chloride

75.67 75.61 75.47 75.29 75.09 74.87 China, and calculate the isosteric heat of water sorption

from the experimental data, providing theoretic basis 86.77 85.92 85.11 84.34 83.62 82.95 chloride for rice drying and storage.

Potassium

Potassium nitrate

a Source: Jayas and Mazza [1].

2. Materials and Methods

humidity (r.h.) levels ranging from 11.3 to 96%. Every

2.1 Materials and Experimental Procedures r.h. at one temperature was triplicated and a total of 135

Thirteen rice (Oryza sativa L.) varieties in China bottles was used in the experiment for five sorption were used in the sorption isotherm experiments. These

isotherms of a rice variety. The temperature of cabinets rice varieties, including three classes of Japonica rice

was often proofed with a standard thermometer ranging and ten classes of Indica rice, were respectively

from 0 to 50 ℃, and controlled to an accuracy of ± collected from eight provinces of the major rice grain

0.5 ℃. The sample of rice seeds (about 4-5 g) was put production regions in China during 2007 to 2009. The

into a small bucket (3 cm diameter × 4 cm length) made rough rice grains used for this study were intact, clean

from copper wire gauze, and hung into the 250 ml and plump. For adsorption experiment, the rice grains

bottle on a copper wire pothook under a rubber plug, were dried to the moisture content (m.c.) of 7-8% wet

just 2-3 cm above saturated salt solutions. The rubber basis (w.b.) at 39.0 ℃ in an oven, and then dehydrated

plug was tightly plugged into the bottle mouth. From by P 2 O 5 solid in a dessicator to below 5% w.b. as being

three weeks after exposing the samples in the saturated samples. For the samples of desorption experiment, the

vapour at 35 ℃, the copper wire buckets with samples rice varieties were re-moisturized from below 5% w.b.

were weighed every other day until the change in mass to the m.c. of 22% w.b., and equilibrated at 4 ℃ for two

between two successive readings was less than 2 mg. weeks.

When the sample was exposed to a lower temperature, The static gravimetric method, with standard

the sample was left several days to equilibrate. saturated salt solutions in Table 1 to maintain constant

However, the rice grains exposed over the saturated vapor pressure [1], was used to obtain nine equilibrium

potassium nitrate solution for 5 to 7 days at higher moisture contents at each of five constant temperatures

temperatures were susceptible to grow molds, and (10, 20, 25, 30, and 35 ℃). Twenty-seven glass bottles

immediately taken out once one seed molded. The with a volume of 250 ml each contained 65 ml salt

moisture content of the sample at this constant stage solution, and were kept in one temperature controlled

was defined to be the equilibrium moisture content cabinet to maintain nine groups of different relative

(EMC) and was determined by an oven method [9].

The Sorption Isosteric Heats of Rough Rice in China

The sample was dried to constant weight under 103.0 quality of the fit. The Eqs. (1)-(4) were used for ± 0.5 ℃ for 22-28 hours. 2 calculating R , RSS, SE, and MRE, respectively.

2.2 Analysis of the Adsorption and Desorption Data ( m i − m 2 pi )

2 (1) The adsorption and desorption EMC data of rough

∑ ( m i − m mi )

rice were fitted to the six moisture sorption isotherm

equations given in Table 2, using the non-linear

RSS = ∑ ( m i − m pi ) 2 (2)

regression procedure in SPSS 13.0 for Windows [10],

which minimizes the sum of squares of deviations

( m − m ) ∑ 2 i pi

(3) between experimental and predicted data in a series of

SE =

( n − 1 ) iterative steps. The goodness-of-fit of each equation

2 m i − m pi was evaluated using determination coefficient (R ),

i = 1 m i (4) residue sum of squares (RSS), the standard error (SE),

MRE =

n and mean relative percentage error (MRE).

where m i is the experimental value ， m pi is the The R 2 was one of the primary criteria for selecting predicated value, m mi is the average of experimental

the best equation to fit the experimental data. In values, and n is the number of observations. The fit of addition to R 2 , the other statistical parameters, MRE as an equation is good enough for practical purposes when

a percentage, RSS and SE were used to determine the

MRE is less than 10% [11].

Table 2 The cited EMC/ERH equations in the study.

Models Equations a

Brunauer-Emmett-Teller (BET)

3 (r.h. < 50%)

( 1 − r . h .) × ( 1 − r . h . + C 3 × r . h .)

× ( 1 − C 1 ) − {[ 2 + 3 × ( 1 − 1 )] 2 − 4 × ( 1 − 3 )} 2

Guggenheim-Anderson-deBoer (GAB)

or M =

C 2 × r . h .) × ( 1 − C 2 × r . h . + 3 × C 2 × r . h .)

r . h . = exp[ − 1 exp( − C 3 × M )] or

Modified Chung-Pfost (MCPE)

Modified Henderson (MHE)

r . h . = 1 − exp[ − C 1 × ( t + C 2 ) × M ]

Modified Oswin (MOE)

C 1 + C × t or

Strohman-Yoerger (STYE) r . h . = exp[ C 1 × exp( − C 2 × M ) × ln( P s ) − C 3 × exp( − C 4 × M )] a r.h. represents moisture content, M is equilibrium moisture content (% dry basis), t is temperature (℃), and P

s is saturated vapor

pressure. C 1 ,C 2 ,C 3 , and C 4 are the coefficients of equations.

18 The Sorption Isosteric Heats of Rough Rice in China

MCPE, MHE, MOE and GAB gave the better fit to the

2.3 Determination of the Isosteric Heat of Sorption experimental data of adsorption and desorption

The total energy required to remove a unit mass of isotherms in a wide range of 11.3 to 96.0% ERH, but water from grain kernels, the differential heat of

the BET equation gave the better fit in the range of 11.3 sorption (h s ), is conveniently partitioned into two

to 49.9% ERH (data not shown). The further components, namely the latent heat of vaporization of

comparisons of the sorption equations with a form of free water (h v ) and the differential heat of wetting (h w ).

rh .. = fMt (,) or M = frht ( . ., ) for twenty-six sets The h v of adsorption and desorption of rice were of isotherm data are given in Table 3. The average

respectively calculated by the following six equations 2 values of R and error parameters (RSS, SE, and MRE) according to Thorpe [7].

for the twenty-six sets of isotherm data were

p s dT ∂ rh .. =+ calculated.

rh .. dP s ∂ T mc ..

For the form of rh .. = fMt (,) , the equations were

h v = 2501.33 2.363 − × t ranked for accuracy in an order: STYE, MCPE, MHE, (6)

MOE, MHE and GAB, but for that of M = frht ( . ., ) , P s =

the order was: BET, MCPE, MHE, MOE, and GAB. dP s

(273.15 + t )

t + 273.15

= However, STYE is four-coefficient, temperature s ×

dT ( t + 273.15) t + 273.15 independent equation and can not be explicitly inverted ∂ r . h .

2 × exp( − C 3 × m . c .)

to give EMC as a function of ERH. MCPE, MHE,

MOE and GAB equations all are three-coefficient, The Eq. (5) enables one to calculate h s /h v , provided

temperature dependent and easily invertible equations dP s /dT and ∂ rh . ./ ∂ T mc .. can be evaluated by Eqs. (8)

(Table 2). Thus, the MCPE with a form of and (9), respectively. The h v of free water in Eq. (6) is

rh .. = fMt (,) , or with a form of M = frht ( . ., ) was dependent on temperature (t, ℃). The saturated vapor

considered to best describe the equilibrium moisture pressure (P s ) can be calculated by Eq. (7). The

data of thirteen rice varieties in a wide range of 11.3 to derivative of r.h. with respect to t, ∂ rh . ./ ∂ T mc .. depends

96.0% ERH, and the best fitted coefficients for both on the sorption isotherm equation used, and the

adsorption and desorption isotherms of rough rice data Modified Chung-Pfost (MCPE) in Eq. (9) is used in

were summarized in Table 4. For MCPE model, the this study.

three coefficients C 1 ,C 2 and C 3 of adsorptive isotherm

3. Results

equation were different from those of desorptive isotherm equation, and there were some difference in

3.1 Fitting of Sorption Equations to Experimental three coefficients C 1 ,C 2 and C 3 of between Japonica Sorption Data

and Indica rice.

The results of nonlinear regression analyses of

3.2 Isosteric Heat of Sorption

fitting the sorption equations to the experimental data of desorption and adsorption isotherms were

The isosteric heat of sorption (h s ) was calculated respectively evaluated with the indices such as

from the Eqs. (6) to (9). The coefficients C 1 ,C 2 , and C 3 correlation coefficient (R 2 ), residue sum of squares

of MCPE equation in Table 4 were used as the (RSS), the standard error (SE), and mean relative

coefficients in Eq. (9). Fig. 1 shows the influence of percentage error (MRE). Of the six commonly used

moisture content on rough rice adsorption and equations, namely BET, GAB, MCPE, MHE, MOE,

desorption isosteric heats, and on the sorption isosteric and STYE (Table 2), five equations such as STYE,

heats of Japonica and Indica rice determined from

The Sorption Isosteric Heats of Rough Rice in China

Table 3 Summary of the results of fitting equations to the data sets of thirteen pairs of rice desorption and adsorption.

Statistical parameters a

Model function

rh .. = fMt (,) MHE

3.65875 BET 0.98096 1.91840 0.10670 2.98630 GAB 0.97031 47.73069 1.13642 8.46685 M = frht ( . ., ) MCPE 0.99225 12.24169 0.29150 3.02528

MHE 0.98675 21.32694 0.50775 4.52040 MOE 0.98031 31.34288 0.74628 6.02336

a The statistical parameter is average of the data sets of thirteen pairs of rough rice desorption and adsorption. R 2 , correlation coefficient; RSS, residue sum of squares; SE, standard error; MRE means relative percentage error.

Table 4 The best fitted coefficients of MCPE for sorption isotherms of rough rice.

Model coefficients

Statistical parameters

Model Data sets a

C C C R 1 2 2 3 RSS SE MRE % Desorption 412.543 35.300 0.181

1.094E-04 1.7654 Adsorption 677.146 110.639 0.184

rh .. = fMt (,) Average 483.486 57.569 0.182 0.9985

1.131E-04 1.6690 (MCPE) Japonica rice 455.064

1.682E-04 2.5580 Indica rice

1.136E-04 1.6934 a Data sets were derivated from the average sorption data of thirteen rice varieties. Desorption, desorption isotherm; Adsorption,

adsorption isotherms; Average, the average values obtained from adsorption and desorption isotherms.

adsorption and desorption isotherms. those of Japonica rice at all moisture contents under a The isosteric heats for both rough rice desorption

constant temperature (Fig. 1B).

and adsorption (Fig. 1A), and for both the sorption of

4. Discussion

Japonica and India rice (Fig. 1B) decreased rapidly with increase in seed moisture content until the

Sun [2] analyzed 17 source sets of rice EMC/ERH moisture content of 20% d.b. was reached, but above

data with four commonly cited models such as MCPE, 20% d.b. they decreased smoothly with increasing

MHE, MOE, and STYE, and considered the STYE as moisture content. At lower moisture contents below

the preferred equation, but three models like MHE, 22.5% d.b., the isosteric heats of both desorption and

MCPE, and MOE were recommended to fit the adsorption of rough rice, and of both sorption of

EMC/ERH data of rough rice by ASABE [5]. In this Japonica and Indica rice under lower temperatures

study, for the form of rh .. = fMt ( ,) , the equations were higher than those under higher temperatures. The

were ranked for accuracy in an order: STYE, MCPE, isosteric heats of rough rice desorption were higher

MHE, MOE, MHE and GAB, but for that of than those of adsorption below 22.5% d.b., but

M = frht ( . ., ) , the order was: BET, MCPE, MHE, thereafter there was no difference found between

MOE, and GAB. The authors regarded MCPE as the desorption and adsorption (Fig. 1A). The sorption

most fitted equation due to it being three coefficients, isosteric heats of Indica rice were slightly higher than

invertible equations. Thus, MCPE was adopted in this

20 The Sorption Isosteric Heats of Rough Rice in China

A Desorption-10℃

above 11% d.b. EMC the isosteric heats of desorption

were lower than those of adsorption. The sorption 4500

Desorption-20℃ Desorption-25℃

isosteric heats of Indica rice were insignificantly higher ion

than those of Japonica rice under all moisture contents or

Adsorption-10℃

s Adsorption-20℃ )

at a constant temperature. The rapid increase in the heat of

Adsorption-25℃

at Adsorption-30℃ /Kg 3500

of sorption at low m.c. might be due to the existence of he (KJ ic

Adsorption-35℃

3000 highly active polar sites on the surface of rice grains, er

ost which were covered with water molecules forming a Is

2500 mono-molecular layer [13]. The decrease in the 2000

isosteric heats with higher amounts of sorbed water can

be quantitatively explained by considering that m.c. (decimal d.b.) sorption initially occurs on the most active available

B sites giving rise to high interaction energy. As these

sites become occupied, sorption occurs on the less ion Japonica-25℃ 3600

3800 Japonica-10℃

Japonica-20℃

active ones, resulting in lower heats of sorption [14]. In pt

Japonica-30℃

low moisture contents, the values of the isosteric heats sor

were higher than the latent heat of vaporization of at J/ 3200

g) of Indica-20℃ K

water, indicating that the energy of binding between

Indica-35℃

ri the water molecules and the sorption sites was higher te

than the energy which holds the molecules of pure Is 2600

os 2800

water together in the liquid phase [15]. At high

moisture contents, there was no significant difference

between the sorption isosteric heat and the latent heat m.c. (decimal d.b.)

Fig. 1 Comparison of adsorption and desorption isosteric

of vaporization of water over the broad range of

heats of rough rice (A), and of the sorption isosteric heats of

moisture contents. In the present study, the heat of

Japonica and Indica rice (B) at different temperatures (℃)

sorption of rough rice grains might approach that of

predicted by the Modified Chung-Pfost equation.

pure water at the moisture content of about 22.5% d.b. study to calculate the rough rice isosteric heat of sorption.

It has been noted that h s /h v was calculated to be The isosteric heats for both rough rice desorption

dependent on temperature, but the dependence was and adsorption, and for both the sorption of Japonica

small [7]. This temperature dependency of h s /h v was and Indica rice, decreased rapidly with an increase in

rather clear in Fig. 1, which might arise from seed moisture content until the m.c. of 20% d.b. was

experimental errors in measuring the sorption isotherm, reached, and thereafter they decreased smoothly with

or from the sample properties such as variety, harvest increasing moisture content. The isosteric heats of

time, pre-treatment, and so on.

rough rice desorption were higher than those of In summary, MCPE was the best fitted equation to adsorption below 22.5% d.b. EMC, but thereafter there

describe the EMC/ERH data of rough rice for thirteen was no significant difference found between desorption

Chinese varieties, and was adopted to calculate the and adsorption. These results show a little difference

isosteric heat of sorption. The isosteric heats of rough from the results reported by Öztekin and Soysal [12]

rice desorption were higher than those of adsorption that the isosteric heats of rice desorption were higher

below 22.5% d.b. EMC, but thereafter there was no than those of adsorption below 11% d.b. EMC, but

difference found between desorption and adsorption.

The Sorption Isosteric Heats of Rough Rice in China

The sorption isosteric heats of Indica rice were slightly [5] ASABE Standards, ASAE D245.5 OCT1995, Moisture higher than those of Japonica rice under all moisture relationships of plant-based agricultural products, American Society of Agricultural Engineers, St. Joseph,

contents at a constant temperature. These results could Michigan, USA, 2006, 49085-49659.

be used for rice drying, storage and aeration. [6] H.A. Iglesias, J. Chirife, P. Viollaz, Thermodynamics of water vapour sorption by sugar beet root, Journal of Food

Acknowledgments

Technology 11 (1976) 91-101. [7] G.R. Thorpe, Physical basis of aeration, in: S. Navarro, R.

The authors gratefully acknowledge t he Scientific Noyes (Eds.), The Mechanics and Physical of Modern Research Foundation for the Returned Overseas

Grain Aeration Management, CRC Press, Boca Raton, Chinese Scholars, State Human Resources and Social

2001, pp. 135-144, 186. [8] X.J. Li, Z.Y. Wei, Z.Y. Cao, Q.Y. Feng, J.S. Wang,

Security Department (CZ1020) and the National Key Equilibrium moisture content and sorption isosteric heats

Technology R&D Program (Project no. of five wheat varieties in China, Journal of Stored 2009BADA0B00-4) for providing financial support.

Products Research 47 (2011) 39-47. The authors also thank Prof. Digvir S. Jayas from the

[9] AOAC, Official Methods of Analysis, 13th edition, Washington DC, Assoc. Off. Anal. Chem., 1980.

University of Manitoba in Canada for his kindly [10] SPSS Inc., SPSS for Windows, Release 13.0.1., SPSS Inc.,

suggestions for the experiment, and Dr. Graham

Chicago, USA, 2006.

Thorpe of Victoria University, Australia for [11] R.J. Aguerre, C. Suarez, P.E. Viollaz, New BET type demonstrating how to calculate the isosteric heat of

multilayer sorption isotherms: Part II. Modelling water sorption in foods, Lebensmittel-Wissenschaft

sorption. und-Technologie 22 (1989) 192-195.

[12] S. Öztekin, Y. Soysal, Comparison of adsorption and

References

desorption isosteric heats for some grains, Agricultural [1] D.S. Jayas, G. Mazza, Equilibrium moisture

Engineering International: the CIGR Journal of Scientific characteristics of safflower seeds, Transactions of the

Research and Development 2 (2000)1-17. ASAE 34 (1991) 2099-2103.

[13] E. Tsami, Net isosteric heat of sorption in dried fruits, [2] D.W. Sun, Comparison and selection of EMC/ERH

Journal of Food Engineering 14 (1991) 327-335. isotherm equations for rice, Journal of Stored Products

[14] N. Wang, J.G. Brennan, Moisture sorption isotherm Research 35 (1999) 249-264.

characteristics of potatoes at four temperatures, Journal of [3] X.J. Li, S.L. Wang, J.S. Wang, Progress in

Food Engineering 14 (1991) 269-282. characterization of equilibrium moisture content in grains,

[15] A.H. Al-Mutaseb, W.A.M. McMinn, T.R.A. Magee, Journal of the Chinese Cereals and Oils Association 24 (11)

Water sorption isotherms of starch powders. Part 2: (2009) 137-145. (in Chinese with English abstract)

Thermodynamic characteristics, Journal of Food [4] C. Chen, R.V. Morey, Comparison of four EMC/ERH

Engineering 62 (2004) 135-142.

equations, Transactions of the ASAE 32 (1989) 983-990.

Journal of Life Sciences 5 (2011) 22-28

Alterations of Antioxidative Enzymes Activities and Induction of Lipid Peroxidation in Germinating Wheat Seeds Subjected to Cadmium Stress

Surjendu Kumar Dey Postgraduate Department of Environmental Science, Fakir Mohan University, Balasore-756020, India

Received: May 13, 2010 / Accepted: August 07, 2010 / Published: January 30, 2011.

Abstract: Germinating wheat (Triticum aestivum L.) seeds were exposed to CdCl 2 (50, 100 and 200 µM) for 48 h and some aspects of oxidative metabolism was assessed in the embryonic tissues. The germination percentage and the soluble protein content of the embryonic tissues were found to decrease with increasing of Cd concentration. There was elevation in superoxide dismutase (SOD) and decline in catalase (CAT) and peroxidase (POX) activities. The increasing of lipid peroxidation levels indicated the prevalence of oxidative stress in the tissues which was probably due to the alteration of antioxidative enzymes activities. The adding of ascorbate,

along with CdCl 2 , has resulted in restoration of the Cd induced decline in CAT activity. Weakening in H 2 O 2 detoxification system seems to be the principal reason behind Cd induced oxidative stress in germinating seeds. Thus, imposition of oxidative stress might be the consequence of cadmium stress and this finding may help in elucidating the mechanisms underlying cadmium mediated toxicity in germinating seeds.

Key words: Ascorbate, cadmium stress, catalase, lipid peroxidation, oxidative stress, peroxidase, superoxide dismutase.

1. Introduction ions [1]. Because of its solubility in water, Cd is absorbed by plants through the root systems along with

Cadmium, a divalent metal chemically similar to essential mineral absorption. The growth and zinc and mercury, is widely distributed in the development have been reported to be affected by lithosphere. It is usually found at quite low exposure of plants to increasing concentrations of concentrations in crystal rocks. This metal is widely cadmium [2, 3]. The antioxidative enzymes like used in metal electroplating, colour pigment superoxide dismutase (SOD), catalase and peroxidases production used in fabrics, textiles, paints etc., are parts of natural defense systems against the manufacture of alloys and Ni-Cd batteries, as plastic deleterious effects of reactive oxygen species (ROS) in stabilizers, production of certain pesticides and many aerobic organisms. Under normal conditions these other anthropogenic activities. All these industrial enzymes, along with other low molecular antioxidants, applications serve as the potential sources of release of scavenge the toxic ROS and protects the cells from the cadmium to the environment. This is a non-essential cascade of oxidative corrosion [4]. When plants are element without known physiological role in plants and subjected to different environmental stresses including animals. Like other heavy metals, its chemical form in heavy metal stress, activities of antioxidative enzymes soil solution is depended on pH and presence of other have been reported to altered, imposing oxidative stress

situation [3, 5-9]. There are many reports about the Corresponding author: Surjendu Kumar Dey, Ph.D.,

research fields: plant physiology, environmental biotechnology, effects of cadmium on antioxidative systems of plants oxidative stress physiology. E-mail: surjendufmu@rediffmail. com.

during growth and developmental stages. But studies

Alterations of Antioxidative Enzymes Activities and Induction of Lipid Peroxidation in Germinating

Wheat Seeds Subjected to Cadmium Stress

on the effect of this metal on the antioxidative trichloroacetic acid (TCA). After overnight storage in efficiency during germination phase are rare. refrigerator, the samples were centrifuged and pellets Germination is an important phase of plant life during

were washed successively with 10% (w/v) TCA, which the juvenile radicles and plumules emerge out of

absolute alcohol, alcohol : chloroform (3:1), alcohol : dormant seeds. Any physiological injury in these

ether (3:1) and finally with ether. The pellets were tender tissues due to exposure to pollutants like heavy

allowed to dry in air for few mins and resuspended with metals can not only affect the seedling growth, may

3 N NaOH for 16 h at 37 ℃. The samples were then also affect the plant growth at later stages. Thus,

centrifuged and the supernatants were collected for studies on the toxic effects of heavy metals during seed

soluble protein estimation, after suitable dilutions, germination are highly essential. Therefore, in this

following the Folin-phenol reagent method of Lowry et study attempts have been made to assess the effect of

al. [10]. The protein content of the tissue was cadmium on the activities of antioxidative enzymes

calculated from the standard curve, drawn with known like SOD, catalase and peroxidase. At the same time,

concentrations of protein (bovine serum albumin). the lipid peroxidation level was also determined to

2.3 Extraction and Assay of Antioxidative Enzymes know the extent of oxidative injury.

The excised embryonic tissues were homogenized

2. Materials and Methods

under ice cold conditions in a mortar and pestle with

2.1 Plant Material, Germination Conditions and Stress extraction buffers (sodium phosphate buffer, 0.05 M, Imposition

pH 7.4 for SOD; sodium phosphate buffer, 0.05 M, pH

7.5 for catalase and peroxidase). The homogenates Wheat (Triticum aestivum L. cv. Sonalika) seeds

were centrifuged at 17,000 × g for 10 min at 0 ℃ and were selected for the uniformity of size and were

surface sterilized with freshly prepared and filtered 3% the resultants supernatants were desalted by passing (w/v) solution of commercial bleaching power through gel filtration columns, packed with pre-soaked (CaOCl

2 ) for 30 min. The seeds were then washed for several times, for another 30 min with double distilled

Sephadex G-25 (fine grade). The eluted fractions

responding to protein test were pooled and used water (ddH

2 O) and were spread over moist filter in Petri dishes for germination. Before spreading the

directly for enzyme assay. Superoxide dismutase (SOD,

EC 1.15.1.1) was assayed by measuring the inhibition seeds, the filter papers in Petri dishes were soaked with

of superoxide-driven nitrite formation from

5 ml solutions of CdCl 2 (50, 100 and 200 µM,

hydroxylamine hydrochloride, as described by Das et

al. [11]. SOD activity was calculated from the value of Petri dish as control. Twenty sterilized seeds were

separately). 100 ml of ddH 2 O was taken in another

V o /V-1, where V o is the absorbance (read at 543 nm) of spread in each Petri dish to maintain uniformity in

the control (without enzyme) and V is absorbance of stress imposition and all the Petri dishes were kept in

the sample (with enzyme). Catalase (CAT, EC 1.11.1.6) dark at 30 ± 2 ℃. After 48 h, the germinating embryos

activity was assayed spectrophotometrically by were excised (discarding the endosperm portion) for

measuring the decreasing rate of H 2 O 2 concentration in different biochemical analysis.

the reaction mixture due to enzyme at 240 nm following the method of Aebi [12]. Catalase activity

2.2 Soluble Protein Extraction and Estimation was calculated by using the extinction coefficient of

40.0 mM -1 cm -1 for H 2 O 2 at 240 nm. Peroxidase (POX, enzyme supernatant (the extraction procedure of which

Buffer soluble protein was precipitated by mixing

EC 1.11.1.7) was assayed using guaiacol and H 2 O 2 as is mentioned below) with equal volume of 20% (w/v)

substrates and by recording the rate of increase in

24 Alterations of Antioxidative Enzymes Activities and Induction of Lipid Peroxidation in Germinating Wheat Seeds Subjected to Cadmium Stress

absorbance due to tetraguaiacol formation at 470 nm, severe consequence on plant growth. In this study, the as described following the method of Kar and

toxic effect of cadmium was detected initially in the Feierabend [13]. The POX activity was calculated

form of inhibition in germination percentage. At 50 µM using the extinction coefficient of 26.6 mM -1 cm -1 due

CdCl 2 , there was 24% germination inhibition which to tetraguaiacol formation.

further increased to 41% at 200 µM in comparison to the control (Fig. 1). At the same time, the soluble

2.4 Extraction and Estimation of Malondialdehyde protein content of the embryonic tissues decreased

The level of lipid peroxidation in the tissues was significantly in a concentration dependent manner and determined by measuring malondialdehyde (MDA), a

at 200 µM, there was almost 65% decline in protein decomposition product of peroxidized polyunsaturated

content than the control one (Fig. 1) which indicated fatty acid component of membrane lipid, using

the probability of alterations in the enzyme levels. thiobarbituric acid (TBA) as the reactive material,

following the method of Heath and Packer [14] and the

concentration of MDA was calculated by using the

extinction coefficient of 155 mM -1 cm -1 for MDA at

2.5 Data Presentation and Statistical Analysis

One unit of SOD activity was defined as the amount

that inhibits the superoxide-driven nitrite formation

from hydroxylamine hydrochloride by 50% under the laboratory conditions. The activities of CAT and POX were expressed in katals (kat), i.e., moles of substrate

consumed or product formed per second. All the

experiments were performed at least for three times

with three replicates in each time. Mean values are presented and the standard deviations are indicated.

FW

The significance of difference between the means was

gg

determined following the LSD test [15]. c

ei n, 10 ot

3. Results and Discussion

e pr

ubl

Germination of seeds is an important phase of plant

S ol 5

growth and development. It is the first exchange interface of the plant life with surrounding medium and therefore, is considered as highly sensitive to

environmental changes [16]. The germination process CdCl 2 , µM starts with the imbibition of seeds which initiates

Fig. 1 Changes in the seed germination percentage (A) and

sequence of events leading to emergence of embryo

soluble protein content of embryos (B) of wheat seeds

exposed to CdCl 2 radicle [17]. During this stage since the tissues are very for 48 h. The values are the mean ± SD of

three independent experiments each with three replicates.

tender and delicate, any exposure to pollutants

The mean values followed by the same letters are not

including toxic heavy metals like cadmium would have

significantly different (P ≤ 0.05; LSD test).

Alterations of Antioxidative Enzymes Activities and Induction of Lipid Peroxidation in Germinating

Wheat Seeds Subjected to Cadmium Stress

prot 28 -1 pr

activ X 5 P O

CdCl 2 , µM

Fig. 2 Changes in the activities of superoxide dismutase (SOD) (A), catalase (CAT) (B), and peroxidase (POX) (C) in germinating wheat embryos exposed to CdCl 2 for 48 h. The values are the mean ± SD of three independent experiments each with three replicates. The mean values followed by the same letters are not significantly different (P ≤ 0.05; LSD test).

Superoxide dismutase is an important antioxidative situation the protection against H 2 O 2 becomes very enzyme that efficiently dismutates superoxide radicals

essential in the cells in order to reduce the Cd induced to O 2 and H 2 O 2 [4]. In this study it was observed that

oxidative damage. Catalase is the important H 2 O 2 with increase in Cd concentrations, there was elevation

metabolizing enzyme and peroxidases are the H 2 O 2 in SOD activity which reached at significant level at

utilizing enzymes in aerobic cells. Catalase 100 µM CdCl 2 (Fig. 2). Thus with the imposition of Cd

decomposes H 2 O 2 to O 2 and H 2 O and thereby acts as an stress there was induction in the SOD activity in the

important sink for H 2 O 2 whereas POXs utilize H 2 O 2 tissues which prevented the build up of superoxide

during their co-oxidation of reduced co-substrates [4]. radicals. Similar increase in SOD activity due to Cd

The activities of both these enzymes declined stress has been reported during early seedling growth in

significantly with increase in Cd concentration in the Brassica napus by Meng et al. [18]. With elevated

germinating medium (Fig. 2) which increased the SOD activity, there would be increase in the

chances of H 2 O 2 accumulation in tissues. Meng et al. accumulation of H 2 O 2 (the dismutation product of SOD

[18] had also reported the decrease in CAT activity due catalysed reaction) in the tissues. Therefore, in such a

to Cd stress in Brassica napus seedlings with slight

26 Alterations of Antioxidative Enzymes Activities and Induction of Lipid Peroxidation in Germinating Wheat Seeds Subjected to Cadmium Stress

increase in the activity at lower concentration of the

metal. But POX activity was increased in their study

ab

which contradicted the findings. In another report of

this laboratory, POX activity was found to decrease in

bc

FW

root tissues and increase in shoot tissues of wheat

. g ol 45

seedlings grown in presence of CdCl 2 [3]. Increased

, nm

POX activity generally indicates the stressful situations, A

D 30

since the cell-wall bound peroxidases are released under such conditions and hence the elevated activity is

usually not attributed to its H 2 O 2 utilization efficacy. In

this study since POX activity has been found to

decreased, its role in H 2 O 2 utilization is totally

CdCl 2 , µM

insignificant under Cd stress. Therefore, with decrease

Fig. 3 Changes in the level of lipid peroxidation in

in the activities of both CAT and POX, the chances of

germinating wheat embryonic tissues exposed to CdCl 2 for

H 48 h. The values are the mean ± SD of three independent

2 O 2 accumulation increased in the tissues which

experiments each with three replicates. The mean values

might impose oxidative stress situation.

followed by the same letters are not significantly different (P

The superoxide radical and H 2 O 2 reacts in presence

≤ 0.05; LSD test).

of transition metal ions forming hydroxyl radicals via one of the mechanisms behind Cd induced toxicity in

Haber-Weiss reaction [19]. In this study even though germinating seeds. This proposition seems apparent

the elevated SOD activity has protected from since the Cd induced decline in CAT activity was

superoxide radicals, the decline in the activities of both restored almost to the control level when an antioxidant

CAT and POX has favoured the accumulation of H 2 O 2 .

(i.e., ascorbate) was added externally to the The hydroxyl radicals are also known to be formed

germinating medium along with 100 µM CdCl 2 (Table from H 2 O 2 in presence of transition metal ions [4].

1). At the same time, the germination percentage was Thus under Cd stress in the germinating tissues, the

also increased in presence of ascorbate (results not probability of formation of hydroxyl radicals can not be

given). Ascorbate is an important antioxidant in ruled out. In aerobic cells, hydroxyl radical is the most

aerobic cells and is known to scavenge superoxide potentially toxic species among the different reactive

radical, hydroxyl radical and singlet oxygen [21]. This oxygen species. The unsaturated fatty acid components

shows the probability of generation of higher amount of membrane lipids are most vulnerable to its attack

of different reactive oxygen species in germinating and are peroxidized in its presence. The result is the

seeds due to Cd stress and since activities of loss of membrane integrity and cellular architecture

antioxidative enzymes were altered, oxidative stress which may ultimately lead to cellular death under

was imposed.

severe case of oxidative stress. The lipid peroxidation Cadmium is a non-essential heavy metal for normal

is, therefore, considered as a good indicator of the plant metabolism. It is known to cause oxidative stress

prevalence of oxidative stress situation in aerobic cells in plants not directly via Fenton or Haber-Weiss

[20]. The increase in lipid peroxidation level in the reaction, but probably by reducing glutathione level

tissues, as reported herein (Fig. 3), indicates that and/or blocking the photosynthetic electron transport

oxidative stress was imposed in the germinating wheat chain [22]. The studies on the effects of Cd on plants

seeds due to exposure to cadmium. have mostly focused on seedlings or adult plant stage

Thus, imposition of oxidative stress situation might and little information is available regarding the oxidative

Alterations of Antioxidative Enzymes Activities and Induction of Lipid Peroxidation in Germinating

Wheat Seeds Subjected to Cadmium Stress

Table 1 Effect of exogenous antioxidant (ascorbate) on the

elucidating the overall mechanisms behind cadmium

protection of CdCl 2 induced decrease in catalase activity in germinating wheat embryonic tissues. Seeds were exposed to

toxicity in germinating seeds.

ascorbate, along with a toxic concentration of CdCl 2 , for

Acknowledgments

48 h.

Catalase activity

Treatment

The author gratefully acknowledge the Head, P.G.

(nkat. mg -1

protein)

Department of Environmental Science, Fakir Mohan CdCl 2 (100 µM)

Control (water)

19.63 a ± 2.30

University, Balasore-756020, India for providing CdCl

13.75 b ± 1.32

2 (100 µM) + Ascorbate (1 mM) 18.67 ± 0.80 necessary facilities to carry out the work.

CdCl 2 (100 µM) + Ascorbate (10 mM) 19.87 a ± 0.67

The results are mean of three independent experiments with

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[12] H.E. Aebi, Catalase, in: H.U. Bergmeyer (Ed.) Methods of

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[18]

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Germination, Marcel Dekker, New York, 1995, pp. 351-395.

Journal of Life Sciences 5 (2011) 29-34

Effect of Se-S Cooperated Application on the Mineral Content and Nutrition Quality of Garlic (Allium Sativum L.)

1 2 2 1 Huanxiu Li 1 , Changquan Wang , Bing Li , Zesheng Yan and Yangxia Zheng 1. Horticulture College, Sichuan Agricultural University, Ya’an 625014, Sichuan, China

2. Resource and Environment College, Sichuan Agricultural University, Ya’an 625014, Sichuan, China

Received: May 19, 2010 / Accepted: August 19, 2010 / Published: January 30, 2011.

Abstract: Eleven nutrition elements and 5 quality elements of garlic (Allium sativum L.) in different Se, S level and their interaction pot experiments were analyzed by atom absorbing spectrophotometer, titration and fixing sulfur method. The mineral elements were analyzed by the Principal Component Analysis and the Factor Analysis on the SPSS 10.0 and three main factors were picked. The results showed that Se, S and Se-S cooperated application enhanced the garlic nutrition quality by increasing mineral nutrition. The garlic Vc was the highest after using higher Se compared middle S level (S 20 mg/kg soil + Se 40 mg/kg soil). Lower Se level compared middle S level (S 40 mg/kg soil + Se 20 mg/kg soil) get the highest garlic abio-Se in all treatments. The garlic organic Se content was the highest after using higher Se compared middle S (S 40 mg/kg soil + Se 40 mg/kg soil). The treatment lower S compared lower Se level get the highest garlic allicin in all treatment. It showed that the fresh eating factor was affected by the element Se. The flavor factor and health care factor were affected by both Se and S.

Key words: Garlic (Allium stativum L.), Se-S cooperated application, mineral content, nutritional quality.

1. Introduction and “Big condyle”disease of mankind in some area of China and self muscle disease and hydropsy are

There is higher nutrition in garlic and garlic is a related with lower Se in living environment. If man

kind of strengthening body food. The fresh bulbs of absorbs relevance Se, there is the effect on curing

garlic are rich of proteins, fats, vitamins, allicin, Ca, and preventing these diseases [3].

P, Fe and Se. It is approved by nowadays study that Garlic is a rich Se food, the content of Se in garlic

the main medical treatment active component in is 20 to 30 times higher than other vegetables [4].

garlic is organic compound with sulfur compounds, The organic Se content and organized rate of Se

active enzymes, selenium compounds and peptides often become the main value index of garlic medical

[1]. It is shown in research that the organic sulfur and health care quality. The main way to supply Se

compounds (OSCs) in garlic could restrain cancer of for mankind is eating garlic. The different Se and

many animals [2]. Selenium (Se) is necessary sulfur level and their cooperated application were

microelement for mankind, and its rich or lack is carried out for studying the nutrition effect of Se and

correlated closely with mankind and animals’ formal sulfur on the garlic and garlic quality. It could

body metabolizing and health. The “Keshan” disease provide the science evidence for the safety, high

These authors contributed equally to this work. quality and high efficient production of rich Se

Corresponding author: Huanxiu Li, Ph.D., Prof., research

garlic.

field: horticulture science. E-mail: hxli62@163.com.

30 Effect of Se-S Cooperated Application on the Mineral Content and Nutrition Quality of Garlic (Allium

Sativum L.)

2. Materials and Methods

After harvesting garlic samples, fresh samples were treated and analyzed for fresh quality, such as Vitamin

2.1 Materials and Treatments

C (Vc) and allicin content. Part of samples were dried The experiment was carried out in the practice

in oven for the following mineral nutrition analysis, the teaching farm in Sichuan Agricultural University. The

contents of N, P, K, Ca, Mg, S, Se, Fe, Mn, Cu, Zn and air-dry soil 10 kg from the farm was loaded in plastic

organic Se in the samples. H 2 SO 4 -H 2 O 2 digest unite pot with diameter of 25 cm and tallness of 30 cm. The

mensuration was used for the content mensuration of N, pH of soil was 6.57 and there was 23.6 g/kg organic

P and K, HNO 3 -HClO 4 digest-atom absorbing material, 1.16 g/kg total N, 0.20 g/kg total P, 13.8 g/kg

spectrophotometer for the Ca, Mg, Fe, Mn, Cu and Zn total K, 2.19 g/kg exchange Ca, 0.06 g/kg exchange

content mensuration, HNO 3 -HClO 4 digest-BaSO 4 Mg, 93 µg/g alkali dispel N, 18 µg/g rapidly available

turbidimetry for S content mensuration, HNO 3 -HClO 4 P, 94 µg/g rapidly available K, 17 µg/g available S,

digest-HCl deoxidize-hydride occurring atom

0.04 µg/g available Se, 29 µg/g available Fe, 24 µg/g absorbing spectrophotometer for Se mensuration [5]. available Mn, 5 µg/g available Fe and 6 µg/g available

The component of Se (abio-Se and organic Se content) Cu in the soil.

was analyzed by Yongqin Wang’s method [7, 8]. The Five garlic plants were planted in each pot. Se came

garlic Vc was analyzed by 2,6-indigo titration. The from Na 2 SeO 3 and S came from sulfur. The treatment

allicin was analyzed by fixing sulfur method [9]. were following, Se0: Se 0 mg/kg soil (control), Se1: Se

All data were stated and analyzed in SPSS 10.0 for

20 mg/kg soil, Se2: Se 40 mg/kg soil, S0: S 0 mg/kg

Window.

soil, S1: S 20 mg/kg soil, S2: S 40 mg/kg soil, S3: S 80

3. Results

mg/kg soil; and the Se compared with S treatments. The total treatments were 12. Each treatment repeated

3.1 The Effect of the Different Se and S Level on the

4 time (pots) and 3 pots’ plants of each treatment were Mineral Nutrition Content of Garlic analyzed. The pots of all treatments were arranged

The effect of the different Se and S level on the randomly in experiment. mineral nutrition content of garlic was shown in Table The N, P and K fertilizer dosage in each treatment

1. It could be made out from the table that the Se

were same, that was N = 133.33 mg/kg soil, P 2 O 5 =

content in garlic of using 20 mg Na 2 SeO 3 /kg soil and

66.67 mg/kg soil, K 2 O = 133.33 mg/kg soil, and

40 mg Na 2 SeO 3 /kg soil was higher 4.0 times and 5.8 N:P 2 O 5 :K 2 O = 1:0.5:1.0. N was from urea (296.29 times than the control (S 0 + Se 0 ). The Zn, Fe and K

mg/kg soil), P from calcium superphosphate (Ca 2 PO 4

content also increased a little, but the S content in garlic 555.58 mg/kg soil), K from KCl (320.00 mg/kg soil). was lower 7% and 8% individually than the control. 70% N was used as base fertilizer and 30% N was used There were no significant difference in the contents of as after fertilizer, all P, K, Se and S fertilizer were used other mineral elements between different Se level and as base fertilizer. control. The garlic Ca, Fe, Mn, Cu, Zn contents

2.2 Sample Assay and Stat Analysis increased when S level increased, but there were no significant increase of N, P, K, S contents in garlic.

The base physics and chemical character of the soil The effect of Se and S combined treatment on the samples were assayed according to the common Mg content in the garlic was not significant different analysis method by the book [5]. The efficiency Se of from the control. Other nutrition elements, especially K,

soil was assayed by 0.5 mol/L NaHCO 3 soakinghy-

S, Fe, Zn and Se content in garlic were significant dride occurring atom absorbing spectrophotometer [6]. higher in the Se and S combined treatment than the

Effect of Se-S Cooperated Application on the Mineral Content and Nutrition Quality of Garlic (Allium

Sativum L.)

control. The garlic Se content of Se and S combined The Se organic rate also increased along with the Se treatment increased significantly than using Se only,

level in the combination enhancing. The treatment S 1 + which increased 54.3% at least and 100% in the highest.

Se 1 get the highest garlic allicin in all treatment, which The highest garlic Se content was shown on 40 mg

was 9.18% higher than the control (treatment S 0 + Se 0 ). Na 2 SeO 3 /kg soil + 80 mg S/kg soil treatment. It

When S level increased to 40 mg /kg soil and more, the

allicin decreased significantly and the lowest allicin absorbing Se, but restrain garlic absorbing S. The Se

indicated that Na 2 SeO 3 significantly promote garlic

was the treatment S 3 + Se 0, which was 9.08% lower and S combined treatment shows the effect of single Se

than the control (treatment S 0 + Se 0 ). The garlic Vc of using, and also remove the negative effect of single S

middle Se compared middle S (treatment S 1 + Se 2 ) was using. The garlic mineral nutrition, especially the P, K,

the highest, which was significantly higher than other S, Ca, Mn, Cu, Fe, Zn and Se content had been

Se compared S treatment. The second higher Vc was improved by using Se and S.

the treatment S 0 + Se 2 and next was the treatment S 1 + Se 1 ,S 1 + Se 0 ,S 0 + Se 1 and S 0 + Se 0 . The garlic Vc of

3.2 The Effect of Different Se and S Level on the the treatment S 2 + Se 0 ,S 2 + Se 1 ,S 2 + Se 2 ,S 3 + Se 0 ,S 3 Garlic Quality Component + Se 1 and S 3 + Se 2 was lower than the control

The abio-Se, organic Se, Se organic rate and allicin (treatment S 0 + Se 0 ). The garlic Vc of the treatment increased significantly when Se and S combined

S 2 +Se 0 was the lowest, which was 1.6% lower than the treatment was carried out. Lower Se level compared

control (Table 2).

middle S level (treatment S2 + Se1) promoted garlic

3.3 Factor Analysis of Garlic Mineral Nutrition and abio-Se increase, which get highest garlic abio-Se in all

Quality Component

treatments. The garlic organic Se increased along with the Se level in the combination enhancing. The garlic

There are relativity among N, P, K, S, Ca, Mg, Fe, organic Se of high Se compared middle S (treatment S2

Mn, Zn, Cu and Se content in the mature garlic. It + Se2) was 6.6 times higher than the treatment S 2 + Se 0 afforded gist for the statistics latent factor model pact

and 12.6 times higher than control (treatment S 0 + Se 0 ).

variable numbers. The factor analysis tool was chosen

Table 1 The nutrition status of the garlic bulbs in the different selenium-sulphur interaction level.

N P K S Ca Mg Fe Mn Cu Zn Se Treatment

(µg/g) S 0 +Se 0 15.721a 4.353a 8.314a 6.384a 0.675a 0.115a 46.432a 8.213a 1.482a 2.824a 1.16a S 0 +Se 1 16.213b 4.541a 9.242b 5.924a 0.734b 0.151c 47.331b 8.251a 1.551a 2.753a 5.82c S 0 +Se 2 16.105b 4.454a 9.173b 5.880a 0.713ab 0.138ab 54.970d 8.322ab 1.683a 3.285b 7.87d

(g/kg)

S 1 +Se 0 16.654bc 4.604ab 10.185bc 8.016cd 0.721b 0.127a 48.405c 8.281a 1.614a 3.364b 1.894b S 1 +Se 1 17.723c 4.705b 10.436c 8.394d 0.772b 0.144b 56.401e 8.407b 1.662a 3.485b 9.094e S 1 +Se 2 17.014c 4.661b 9.723bc 7.622c 0.767b 0.132ab 64.132f 8.324ab 1.621a 3.593bc 10.462f S 2 +Se 0 16.931c 4.736b 9.923bc 6.981ab 0.767b 0.115a 90.531i 9.583c 2.491b 4.191c 1.791b S 2 +Se 1 15.662a 4.764b 10.972c 6.765ab 0.905d 0.103a 76.975h 9.557c 2.552b 8.932e 10.743f

S 2 +Se 2 16.445b 4.850c 10.567c 7.531c 0.884c 0.140b 75.447g 9.694c 2.655bc 7.586d 11.051fg S 3 +Se 0 17.065c 4.764b 9.955bc 7.074b 0.763b 0.102a 99.177j 9.604c 2.592b 4.506c 1.832b S 3 +Se 1 15.624a 4.743b 11.522d 7.725c 0.961d 0.114a 75.005g 9.645c 2.731bc 8.792e 11.681g

S 3 +Se 2 16.332b 4.881c 10.781c 7.423bc 1.025d 0.131ab 76.332h 9.873d 2.964c 7.634d 11.964g The data was the average of the thrice-repeated test. Significant at the 0.05 probability levels respectively named by the small letters

(n = 36).

32 Effect of Se-S Cooperated Application on the Mineral Content and Nutrition Quality of Garlic (Allium

Sativum L.)

Table 2 The effect of the different selenium-sulphur interaction using on the quality components of the garlic bulbs.

Treatment Abio-Se (µg/g)

Vc (µg/g F) S 0 + Se 0 0.312

Organic Se (µg/g)

Ratio of Org. Se (%)

Allicin (g/kg F)

Aa 72.57 Aa 28.64 Bc 79.49 Bb S 0 + Se 1 0.331

Ab 0.854

Cc 94.32 Cc 29.77 Cc 80.38 Bc S 0 + Se 2 0.344

B bc

Dd 95.79 Cd 29.17 Cc 81.16 Cd S 1 + Se 0 0.293

B bc

Bb 84.25 Bb 29.22 Cc 78.45 Aa S 1 + Se 1 0.391

Aa 1.594

De 95.62 Cc 31.27 Dd 80.56 Bc S 1 + Se 2 0.354

Dd 8.694

Ef 96.60 Dd 31.12 Dd 81.33 Cd S 2 + Se 0 0.294

C cd

Bb 83.89 Bb 27.06 Ab 78.18 Aa S 2 + Se 1 0.335

Aa 1.514

Ef 96.95 De 26.78 Ab 78.76 Aa S 2 + Se 2 0.347

B bc

Fg 97.23 Ee 27.23 Ab 79.03 A ab S 3 + Se 0 0.292

BC c

Bb 83.75 Bb 26.06 Aa 78.46 Aa S 3 + Se 1 0.327

Aa 1.547

Fg 97.25 Ee 26.70 Ab 78.91 A ab S 3 + Se 2 0.366

Bb 11.364

79.15 A ab Significant at the 0.05 and 0.01 probability levels respectively named by the small and capital letters, n = 36.

C cd

Fg 97.45 Ee 26.55 A ab

in the analysis menu of SPSS statistics software. The

Table 3 The picked and analyzed mineral elements and

garlic mineral nutrition factors were picked up by using

factors of garlic bulbs.

Principal Component Analysis (PCA). The mineral

Factor loading

Common factor

Mineral elements

X 1 X 2 X 3 nutrition factors (X1, X2, X3) were picked up variances

0.102 0.103 0.863 according to the eigenvalue which was bigger than 1.0.

S 0.912

0.0882 0.353 0.914 The factor load was treated by the average orthogonal

N 0.891

0.244 0.191 0.855 rotation method in order to explain common factors.

P 0.872

0.143 0.362 0.784 After holding factor value, the common factors became

0.943 0.101 0.931 new variable (twain times variable) for further analysis Se -0.0335 0.913 0.245 0.911 (Table 3).

Fe 0.112

0.855 0.0185 0.885 The X1, X2 and X3 factors covered 83.09% square

Zn 0.314

Cu -0.0385 0.332 0.831 0.803 difference of 11 garlic mineral nutrition element

0.0465 0.761 0.765 Mg 0.588 -0.0605 0.613 variance (Table 3). They offered 37.34%, 26.01%, 0.724

Mn 0.432

2.843 2.173 19.74% square difference individually. The common

Eigenvalues 4.221

26.01 19.74 actor variances of 10 nutrition elements were bigger

Square variance % 37.34

than 75%, only Mg common factor variance was Se and Zn. The third factor X3 covered 19.74% square smaller than 75%. All common factor variances of 11

difference and it had higher positive loading to the Cu, nutrition elements were bigger than 70%. It showed

Mn and Mg.

that the model had higher trust in explaining factor There was obvious linear correlation between garlic variance relationship. The factor loading indicated

nutrition quality and garlic element factors (X1, X2 and relationship between formerly mineral nutrition X3). After the beeline model of their relationship was element variances and the factors. If according to the

estimated, it was found that there was significant factor loading which was bigger than 0.5, the first

beeline relation between allicin and mineral main factor X1 covered 37.34% square difference and it had 2 factor X1 (Y = 28.721 + 0.745X

1 ,R = 0.631, F = higher positive loading to the S, N, P, K, Ca and Mg.

23.514**), organic Se and mineral main factor X2 (Y = The second factor X2 covered 26.01% square 2 7.312 + 3.786X

2 + 1.188X 3 ,R = 0.812, F = difference and it had higher positive loading to the Fe,

88.151**), abio-Se and mineral main factor X2 and X3

Effect of Se-S Cooperated Application on the Mineral Content and Nutrition Quality of Garlic (Allium

Sativum L.)

(Y = 0.445 + 0.0201X 2

tobacco by FA (Factor Analysis) and had a better result. 9.251**), ratio of organic Se and mineral main factor

3 + 0.0156X 2 ,R = 0.503, F =

The authors estimated garlic mineral nutrition element X2 and X3 (Y = 88.645 + 6.103X 2

by using PCA (Principal Component Analysis) and FA 0.732, F = 25.523**), vitamin C and mineral main

2 + 3.544X 3 ,R =

data pact technique and set the 11 nutrition elements (N, factor X3 (Y = 78.432+0.431X 2

P, K, S, Ca, Mg, Fe, Mn, Cu, Zn, Se) off three nutrition 8.732*). Therefore the authors gave X1 a name flavor

3 ,R = 0.311, F =

factors (X1, X2, X3) which were totally inconnected factor which was closely related with allicin and it had

each other. It was entirely viable that using nutrition the positive relationship to the element S, N, P, K, Ca,

quality element named main mineral nutrition factors. Mg; named X2 health care factor which was closely

They contained most data information (75%) and had related with organic Se , abio-Se and ratio of organic Se

predigested complicated data processing. and it had the positive relationship to the element Fe,

4.2 Nutrition Effect of Se and S Single Using Se, Zn; named X3 fresh eating factor which was closely

related with Vitamin C and it had the positive The square variable analysis of mineral factor score relationship to the element Cu, Mn and Mg.

showed that the mineral nutrition factors were significantly affected by single using Se or S. When

3.4 The Effect of Se-S Cooperating on the Garlic single using Se, the contents of N, K, Ca, Mg, Fe, Zn, Nutrition Quality Se in garlics were increased significantly as the

Using Se only had the most significantly effect on concentration of Se solution, but the contents of P, S, the flavor factor (X1), health care factor (X2), fresh

Mn, Cu were not increased significantly. It showed that eating factor (X3), but single using S had the most

single using Se are good for N, K, Ca, Fe, Zn, Se significantly effect on the flavor factor (X1) and health

element accumulation in garlic. When single using S, care factor (X2). Se-S cooperating using had the most

the contents of Fe, Mn, Cu, Zn in garlic were increased significantly effect on the fresh eating factor (X3). It

significantly along with the concentration of S solution, showed that the fresh eating factor was affected by the

but the contents of N, P, K, S, Ca, Mg were not element Se. The flavor factor and health care factor

increased significantly. It showed that single using S is were affected by both Se and S (Table 4).

good for Fe, Mn, Cu, Zn element accumulation in garlic, but not good for N, P, K, S, Ca, Mg element

4. Discussion

accumulation.

4.1 Application of PCA-FA Data Pact Technique on

4.3 Nutrition Effect of Se-S Cooperated Using Plant Nutrition Evaluation

Se and S cooperating using only increased the Huai Cao [10] analyzed the mineral nutrition of function of single using Se or S. Using Se or S or Se

Table 4 The variance and mean square analysis for the

and S cooperating using could increase the edible

effect of the Se-S interaction on mineral factor scores.

quality, remedy quality and flavor quality of garlic by

improving garlic mineral nutrition. Source DF Flavor factor Healthy care Fresh eating

Mean square

There is close relation between Se and S content in S3 17.450** 2.819** 0.0554

(X1)

factor (X2)

factor (X3)

plants. If the plant has the higher organic Se, it contains Se 2 1.531** 6.421** 2.787**

lower S. The phenomena shows on soybean, tomato S×Se 6

and bean [11]. The element Se and S are similar in Error 22 0.102

** shows significant at the 0.01 probability levels respectively, many chemical characters, which decide their

n = 36. similitude of nutrition characters. Se could replace

34 Effect of Se-S Cooperated Application on the Mineral Content and Nutrition Quality of Garlic (Allium Sativum L.)

some S by exchange in the plants and be absorbed by [2] D.M. Liu, X.M. An, Study survey on the drug activity of plants. organic sulfur compounds (OSCs) in garlic, Journal of Chinese Clinic Medicine Research 11 (16) (2005)

Yongqin Wang [7] sprayed Na 2 SeO 3 on garlic and

2365-2366.

found that the garlic production increased firstly and [3] Y.J. Liu, X. Tan, J.G. Liu, H.J. Zhao, X.L. Wang, Effect then decreased along with the Se solution

of selenium-enriched malt on the microvessel density of

matrix and proliferation of tumor cells in hepatoma, concentration increasing (10, 100, 1,000 mg·L ). The Journal of Nanjing Agricultural University 32 (3) (2009) Se content in garlic also increased, but the abio Se

110-114.

transformed into organic Se decreased. It showed that [4] Y.X. Duan, T.Z. Fu, J.R. Fu, Studies on the biological the garlic absorbed Se was mainly affected by the Se

accumulation and antioxidation of Selenium in garlic (Allium sativum L.), Acta Horticulturae Sinica 24 (4)

content in soil or using content. In the experiment, the

(1997) 343-34.

authors added S in applying Se. The result showed that [5] Chinese Pedology Institute, Soil Agricultural Chemical the cooperation using of Se and S nutrition improved

Assay Technique, Chinese Agricultural Science and the garlic allicin and Vitamin C content, increased the

Technology Publishing Company Press, Beijing, China, 1999, p. 638.

ratio of organic Se. The garlic quality increased. It [6] J.G. Qu, B.X. Xu, S.C. Gong, Study on speciation

offers a new approach for high quality, high effect and distribution and availability of Selenium in different soils safe production of rich Se garlic.

of shanghai, Acta Pedologica Sinica 35 (3) (1998) 398-403.

Acknowledgments

[7] Y.Q. Wang, J.S. Cao, J.H. Li, M. Zhao, G.F. Zhao, Effect of Selenium application on the yield of garlic and its This work was supported by Chinese National

selenium content, Acta Horticulturae Sinica 28 (5) (2001) Science and Technology Ministry item “Modern

425-429.

Agricultural Technique Study of Earthquake Rebuild [8] W.K. Huang, Food Test and Analysis, Chinese Light Industry Publishing Company, Beijing, China, 2000, pp.

in An’xian of Sichuan Province” (Item number:

96-98.

2008BAK51B02. Item time: 2008-2010) and Sichuan [9] Z.F. He, D.Q. Zhang, Health Protection Chemistry and Agricultural University’s fund (Item number:

Test Technique, Chinese Light Industry Publishing SAU003305, time: 2008-2010). Company Press, Beijing, China, 1998, p. 123. [10] H. Cao, X.L. Zhang, S.X. Liu, L.Z. He, J.Y. Chen, Y.Y.

References Fan, Factor analysis of mineral element distribution of

flue-cured tobacco, Plant Nutrition and Fertilizer Science [1] C. Ip, M. Birringer, E. Block, M. Kotrebai, J.F. Tyson, P.C.

7 (3) (2001) 318-324.

Uden, et al., Chemical speciation influences comparative [11] Y.H. Zuo, Advances in the studies on forms of selenium in activity of selenium-enriched garlic and yeast in mammary

environment and in plants, Chinese bulletin of Botany 16 cancer prevention, Journal of Agricultural and Food

(4) (1999) 378-380.

Chemistry 48 (6) (2000) 2062-2070.

Journal of Life Sciences 5 (2011) 35-38

Effect of Deficit Irrigation at Different Growth Stages on Wheat Growth and Yield

Seyed Abdolreza Kazemeini and Mohsen Edalat Crop Production and Plant Breeding Dept., College of Agriculture, Shiraz University, Shiraz, Iran

Received: August 01, 2010 / Accepted: October 12, 2010 / Published: January 30, 2011.

Abstract: In order to evaluate the effect of deficit irrigation at various growth stages on wheat growth and yield, a field experiment was conducted in the 2008-2009 growing season based on the completely randomized block with four replications at the experimental farm of the College of Agriculture, Shiraz University, Shiraz, Iran, located at Badjgah. Treatments involved 13 irrigation regimes which applied in stem elongation, heading and grain filling growth stages. The highest seed yield (4,333 kg·ha -1 ) and the lowest ones (1,377 kg·ha -1 ) were obtained from T1 (100% field capacity (FC) in all growth stages) and T13 (50% FC in all growth stages), respectively. With limitation in water amount seed yield was diminished, but this trend was not significant at T4 (100%, 100% and 50% FC) and T11

(100%, 100% and 75% FC). Stepwise regression results revealed that, seed number per spike had the largest contribution (partial R 2 = 0.72) in seed yield variation. Also at T1, T4, and T11 treatments (well water treatments) as well as T13 (sever stress) head number m -2

contribute most in seed yield determination (partial R 2 = 0.96). It can be concluded that water shortage during the grain filling period and its allocation to the other consecutive crop, can increase crop production in southern regions of Iran.

Key words: Deficit irrigation, yield components, wheat, stepwise regression.

1. Introduction be much less than when water stress occurs during flowering period [3]. Appropriate degree of regulated

Water stress is the main factor limiting crop deficit irrigation at the middle vegetative growth period production in dry regions. Deficit irrigation (DI) has (jointing), late vegetative period (booting), early been widely investigated as a valuable and sustainable reproductive period (heading), and late reproductive production strategy in this area. Research results show periods (end of filling or filling and maturity) could that DI can lead in increasing water productivity for result in high grain yield, total biomass, water use various crops without causing severe yield reductions efficiency, water supply use efficiency, harvest index, with occasionally minor yield reductions. Nevertheless, and better yield components in spring wheat in an arid the quality of the yield (e.g., grain size) tends to remain

environment [1].

unchanged or even superior to rain-fed or FI cultivation The objectives of this study were to determine the [1], but a certain minimum amount of seasonal relationship among grain yield and the optimum moisture must be guaranteed. DI requires precise controlled soil water deficit levels and irrigation water knowledge of crop response to drought stress, as at different growth stages in southern Iran. drought tolerance varies considerably by genotype and

phenological stage [2].

2. Materials and Methods

Water stress during growth may reduce yield as

A field experiment was conducted during 2008-2009 compared with full irrigation, but the reduction can still growing season in a silty clay loam soil at the

Corresponding author: Seyed Abdolreza Kazemeini, Ph.D., Experimental Research Center (Badjgah), Shiraz Assi. Prof., research field: crop production. E-mail: kazemin@

University (52°46 ′E, 29°50′N and 1,810 m). The shirazu.ac.ir.

36 Effect of Deficit Irrigation at Different Growth Stages on Wheat Growth and Yield

experimental plots were arranged in a randomized central two rows of each plot were harvested in 15 June complete block design with three replications for each

to measure total dry mater, head number per area and treatment. The treatments included 13 levels of

seed yield. The experimental data were statistically irrigation (Table 1). Mean monthly temperatures and

analyzed for variance using the SAS software. When rainfall rate of the region and some soil characteristics

analysis of variance showed significant treatment are shown in Tables 2 and 3. The soil water content was

effect, LSD Test was applied to compare the means at monitored in each plot using the gravimetric method at

P < 0.05.

30 cm intervals down to 90 cm.

3. Results and Discussion

Time-volume technique [4] is an irrigation technique in which irrigation water is applied by polyethylene

Results showed that the highest and the lowest seed pipes set in each plot and the time of irrigation of each

yields (respectively 4,333 kg·ha -1 and 1,377 kg·ha -1 ) plot was calibrated by a timer and a standard container.

were obtained from T1 (100% FC in all growth stages) Irrigation water amount of each plot (measured by

and T13 (50% FC in all growth stages) respectively. gravimetric method) was converted to time (min) and

Among yield components seed number per spike, applied. Land preparation included plowing, disking

1,000 seed weight and head number per m 2 had the and ridging the plots (3 × 3 m). The fertilizers

maximum values in T1 and the minimum values in T13. consisted of 100 kg triple super phosphate and 100

With limitation in water amount seed yield was kg·ha -1 urea as preplant and 150 kg urea·ha -1 as

decreased, but this trend was not significant in T4 topdressing at 6-leaf growth stage.

(100%, 100% and 50% FC) and T11 (100%, 100% and Weeds chemically controlled by Puma super (1 l·ha -1 )

75% FC).

and Granstar (30 g·ha -1 ) as post emergence application. Results of stepwise regression showed a significant The wheat seeds (var. Shiraz) were sown 5 cm deep

grouping among treatments. Seven groups were and in rows of 20 cm apart by a planter in

distinguished: group 1 (T1, T11 and T4), group 2 (T10 mid-November. At the end of the growing season (15

and T3), group 3 (T12), group 4 (T8 and T6), group 5 June), each plot was harvested for biomass and grain

(T5, T9 and T7), group 6 (T2) and group 7 (T13). yield. Yield components such as thousand-grain weight,

Stepwise regression results revealed that, in group 1 seed number, were measured on 10 plants per plot. The

(well water treatments), seed number per spike had the largest role (partial R 2

Table 1 Deficit irrigation treatments at different growth

= 0.72) in seed yield variation.

stages.

As in cereal, seed number is determined before Treatment

Stem elongation

flowering stage, so sufficient irrigation in this stage can %

Heading

Grain filling

T1 100

be an influential determinant factor in seed yield. Also T2 50

in group 2 seed number per spike had the largest role T3 100

(partial R 2 = 0.62) in seed yield variation. On the other T4 100

hand in group 3, head number per m -2 had the most T5 50

T6 75 100 50 important role in crop yield (R = 0.91). It appears that T7 100

application of mild deficit irrigation before grain filling T8 75

75 75 period can stimulate tiller formation and the tillers T9 50

100 50 became fertile when well water irrigation was used at T10 75

grain filling (Table 5).

In group 4 seed number per spike and 1,000 seed T13 50

weight had the largest role (partial R 2 = 0.85 and 0.098

Effect of Deficit Irrigation at Different Growth Stages on Wheat Growth and Yield

Table 2 Some chemical and physical properties of experimental site soil.

pH Ec N P

FC Bulk density (µmos/s) (%)

(%) (g·cm -3 ) 7.83 2.61 0.091

Table 3 Mean monthly temperature and rainfall in Table 4 Effect of different irrigation treatments on yield 2008-2009 growing season.

and yield components.

Month Max. temperature Min. temperature Rainfall

Biological Seed

(mm)

Treatment yield

yield

Seed Head Seed no./m 2 no./spike

October 26.1 4.2 30.0 (kg·ha -1 ) (kg·ha -1 ) weight November 17.4

May 30.2 9.0 0.0 T 7 5,588.0

29.3 364.0 23.5 respectively) in seed yield formation. In group 5, head

number per m 2 (partial R 2 = 0.72), biological yield

30.5 456.0 (partial R 24.4 = 0.132) and seed number per spike (partial

2.2 27.7 1.8 seed yield variations. As shown in Table 5, 1,000 seed

R = 0.108) were the most important components in LSD (5%) 297.0

2 Table 5 Summary of Stepwise Selection for different

weight (partial R = 1.00) had the greatest effect on

significant groups.

seed yield in group 6. Also in group 13 (sever stress)

R-square R-square value head number m Pr(F) had the most important role on seed

Significant Variable

Partial Model F

2 1 Seed yield production (partial R no./spike 0.7194 0.7194 17.95 0.0039 = 0.96). It can be 2 Seed no./spike 0.6176 0.6176 6.46 0.0639

concluded that decreased water applied amount in the Head no./m 2 0.9173 0.9173 44.38 0.0026 grain filling period and its allocation to the other

Seed no./spike 0.0827 1.0000 5.23 <.0001 consecutive crop, can increase crop production in

Seed no./spike 0.8503 0.8503 22.72 0.0089

southern regions of Iran (Table 5). 1000 seed weight 0.0988 0.9491 5.82 0.0949 Head no./m 2 0.7188 0.7188 4.48 0.0503

4. Conclusion

5 Biological yield 0.1348 0.8536 0.70 0.4159 Seed no./spike 0.1084 0.9621 2.38 0.1452

In this study, the authors evaluated the effect of 6 1000 seed weight 0.9500 0.9500 13.48 <.0001 deficit irrigation at various growth stages on wheat

7 Head no./m 2 0.9616 0.9616 25.04 0.1256 growth and yield. The highest seed yield (4,333 kg·ha -1 )

Grouping: group 1 (T1, T11, T4); group 2 (T10, T3); group 3

and the lowest ones (1,377 kg·ha (T12); group 4 (T8, T6); group 5 (T5, T9, T7); group 6 (T2); ) were obtained from

group 7 (T13).

T1 (100% FC in all growth stages) and T13 (50% FC in yield variation. Also at T1, T4, and T11 treatments all growth stages) respectively. With limitation in

water amount seed yield was diminished, but this trend (well water treatments) as well as T13 (sever stress) head number m was not significant at T4 (100%, 100% and 50% FC) -2 contribute most in seed yield and T11 (100%, 100% and 75% FC). Stepwise 2 determination (partial R = 0.96). It can be concluded

regression results revealed that, seed number per spike that water shortage during the grain filling period and had the largest contribution (partial R 2 = 0.72) in seed

its allocation to the other consecutive crop, can

38 Effect of Deficit Irrigation at Different Growth Stages on Wheat Growth and Yield

increase crop production in southern regions of Iran. strategy to maximize crop water productivity in dry areas, Agr. Water Manage 96 (2009) 1275-1284.

References

[3]

E. Tolga, D. Lokman, Yield response of sunflower to water stress under Tekirdag conditions, Helia. 26 (38)

[1] B. Zhang, F.M. Li, G. Huang, Z.Y. Cheng, Y. Zhang, Yield

(2003) 149-158.

performance of spring wheat improved by regulated deficit [4] D.W. Grimes, H. Yamada, S.W. Hughes, Climate- irrigation in an arid area, Agr. Water Manage 79 (2006) 28-42.

normalized cotton leaf water potentials for irrigation [2] S. Geerts, D. Raes, Deficit irrigation as an on-farm

scheduling, Agric. Water Manage 12 (1987) 293-304.

Journal of Life Sciences 5 (2011) 39-47

Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan

Farzana Perveen, Sardar Azher Mehmood, Shabbir Ahmed and Zia Ur Reman

Department of Zoology, Hazara University, Garden Campus, Mansehra-21300, Pakistan

Received: November 10, 2010 / Accepted: November 29, 2010 / Published: January 30, 2011.

Abstract:

A study was aimed to determine the biological status of the Indus river dolphin, Platanista minor Owen in Indus river with respect to distribution, threats and protection throughout its range in Northern Pakistan. For this purpose, a survey was conducted in an area of 103.5 km including the Indus river and its tributaries in March 2009 for estimating its distribution and population size using both direct observations of the dolphin as well as indirect evidences from its signs. The best group size was estimated with an abundance of 54 dolphins. Approximately 53.7% (1.07 dolphin/km) of the dolphin-population occurred in 27 km from Miran onward to Ramak, however, 38.9% (0.46 dolphin/km) occurred in 46 km from Dera Ismail (D.I.) Khan bridge to Miran, moreover, 7.4% (0.13 dolphin/km) occurred in 30.5 km from Saggu, near Meetapur-village to D.I. Khan bridge in the Indus river. The highest density was found between Miran and Ramk. A pronounced increase in dolphin encounter rate and abundance were observed in a downstream-direction. Threats to dolphins include too much vessel-traffic, disturbance by the crane, duck-hunters, and no effective-protection. Dolphins are particularly vulnerable to the high levels of anthropogenic activities because of their restricted habitat. Threats vary geographically in their importance, but generally include accidental killing during fishing-operations, habitat-loss and population-fragmentation from water development. Deliberate killing for dolphin products also threatens the dolphins. Potential for protections and possibilities for dolphin eco-tourism measures must be taken.

Key words: Dolphin, endangered or critically endangered, Indus river, Northern Pakistan, protection, population.

1. Introduction forest located between Guddu and Sukkur barrages [1-4]. All the three Asian species of freshwater

The Indus river dolphin, Platanista minor Owen is dolphins are classified as endangered or critically endemic to Pakistan and occurs only in the Indus river endangered. Listed in order of the most to least system. The Indus river has five main tributaries, the threatened, are the baiji, Lipotes vexillifer Miller in Jhelum, Sutlej, Chenab, Ravi and Beas Rivers. These River Yangtze of China (population: a few tens), the rivers merge with one another to form the Panjnad, bhulan, Platanista minor (Roxburgh) in Indus river of which then joins the Indus main stem. The Indus Pakistan (population: a few hundreds), and the Susus, leaves the Himalayan foothills and enters the plains at Platanista gangetica (Roxburgh), population about Kalabagh, 3 km upstream of Jinnah Barrage. From 2,500. The fourth freshwater species, the boto, Inia Kalabagh, it flows at a gentle gradient (averaging geoffrensis (de Blainville) is found in the Amazon

13 cm/km), primarily, for approximately 1,600 km, river system in South America, population of which is to its confluence with the Arabian Sea. It runs through estimated to be about 5,000 [5-8]. semi-desert and irrigated agricultural land, as well as The typical Indus river dolphin is having a sleek some small remnant areas of native riverine scrub fusiform body, the caudal region being laterally

Correspondence author: Farzana Perveen, Ph.D., research compressed and very slim. It differs from other fields: conservation and wild life management, entomology,

dolphins in the broad spade-shaped flippers and toxicology, biotechnology and genetic engineering. E-mail:

farzana_san@hotmail.com. rostrum or beak, which is relatively long, and very

40 Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan

slender. In colour, the dolphin is pinkish or purplish influences, fourteen species of dolphins are considered grey-brown, being only slightly paler ventrally [9-12].

to be in danger of extinction according to the Indus dolphin, though blind, locates and identifies

Endangered Species Act. Two species of river dolphins objects and prey (food) through sonar system [13-16]

are currently the most endangered [23]; therefore, their or echo-location is especially well-developed. Its

protection and conservation are obligatory. The eyes have become non-functional, lacking any lens,

objectives of the current research are to obtain though there is an extremely small optic nerve [17].

information about its distribution in an area of 103.5 Thus they never cease to emit continuous sound

km including the Indus river and its tributaries in pulses having the frequency of 01-90 sec and these

Northern, to identify threats to its population and to sound bursts are emitted irregularly at intervals of

suggest recommendations for its protection. 01-60 sec [18]. There is sexual dimorphism in this

2. Materials and Methods

dolphin with the males having a markedly shorter rostrum and generally being slightly smaller in body

2.1 Methodology of Survey

length [19]. In the middle of the March, males start Research has been conducted at Department of chasing females by running after them and knocking Zoology, Kohat University of Science and Technology, their bodies with beaks. Mating takes place often in Kohat, Northern Pakistan. The vessel-based dolphin shallow water in late April and May. During mating, survey was conducted in March, 2009. The survey pairs swim parallel to each other and speedily come was covered with an area of 103.5 km along the Indus close to each other touching their ventral sides [16]. river from Saggu (near Meetapur village) (N32° 01.7; Two or three different males are reported to circle E71° 02.7) to Ramak (place near Indus river) (N31° around during copulation and the female 19.4; E70° 47.3). A camp was established next to a subsequently mates with one or two other males [20]. bund and ferry crossing which was located at N31° The places where dolphins found in groups are 25.858; E70° 46.674. Survey methods followed those declared as “Dolphin Schools”, and there are about 17 described by Smith and Reeves [24]. Observers Dolphin Schools in between Guddu and Sukkur consisted of D.I. Khan Wildlife Department staff barrages, where one can have a good chance to watch were trained in dolphin survey methods. Survey was and wonder the dolphin diving’s at closer distance of conducted from oar-powered traditional wooden no more than 50-100 feet. Animal is social but good vessels that were easily available on all the sections of in his natural habitat and any attempt to play with the Indus with low cost on rent which was traveling at animal out of his native home, will be at the cost of its 5-7 km/h in a downstream direction. The vessel life [16]. Dolphins have little sleep rather than surveyed was in a single transect, followed the keep-on moving all the time in search of food [16]. deepest channel and moved from bank to bank as the The marked seasonal change in dolphin distribution is channel meandered. Observers surveyed with the due to fluctuations in water levels [4]. During the dry naked eye and Nikon 7 × 50 binoculars from a season from October to April, many dolphins leave viewing platform approximately 3 m above the water the tributaries of the Ganges-Brahmaputra systems surface. Surveys were conducted using three forward and congregate in the main channels, only to return to observers. Left observer surveyed the water from in the tributaries the following rainy season [21]. Some front of the vessel to 90º from the bow on left side. individuals, mainly juveniles, remain in the Central observer surveyed the river directly in front tributaries throughout the dry season [22]. of the vessel and 45º either side of the bow. Right As a result of whaling and other human and natural observer surveyed the water from in front of the

Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan

vessel to 90º from the bow on the right side. One rear sightings were confirmed by a second observer. observer faced the rear of the vessel and search for

Group sizes were evaluated with a best, high and low dolphins missed by the main survey team and also

estimate of numbers to account for the degree of assisted the primary team in group size estimation

uncertainty. A low and best estimate of zero was used and group tracking. A data recorder was responsible

if the sighting was unconfirmed or if there was a for filling the data sheets and using the GPS (Global

possibility that the dolphin was following the vessel Positioning System). Team members rotated positions

and might have already been counted [25]. Good every 30 min and received a rest period to maintain

coordination between all the observers, especially alertness. While observers focused on obtaining an

forward and rear was essential to obtain an accurate accurate group size estimate.

estimate of group size.

2.2 Collection of Data

2.3 Sightings

The survey data form was used for collecting data Dolphin groups were generally sighted on survey effort. All boxes on each line were filled on

downstream of the survey vessel. When a group was the form on: a) beginning of each day; b) at the end of

sighted, the “detection” location was recorded each day; and c) when the observers changed position

through GPS, a second “exact” location was recorded or when survey stopped because of a dolphin sighting.

when the vessel was located in the centre or Environmental conditions were also recorded at the

perpendicular to the group. When the vessel was a beginning, end of each period of survey, when

beam of the group, channel width was recorded by observers rotated positions, and when conditions

adding the distance from the vessel to each bank. The changed. The effect of wind on the river surface was

probability of double counting due to their movement evaluated according to the following scale: 0: water

from surveyed to un-surveyed reached overnight, was surface glassy; 1: ripples without crests; 2: small

considered to be balanced by the probability that an wavelets with crests but no white-caps; 3: large

equal number of dolphins were missed due to their wavelets with scattered white-caps; 4: small waves

movements in the opposite direction. with fairly frequent white-caps. Visibility was

2.4 Data Analysis

assessed as: 0: clear; 1: visibility less than 2 km;

2: visibility less than 1 km. When viewing conditions The group size was totaled to give high and low deteriorated to river surface state “3” or visibility

estimates of overall abundance. Abundance always code “2”, survey was postponed until conditions

refers to calculate the sum of the best group size improved. Garmin 3 + GPS units were used to record

estimates. For determination of the mean and the survey track and location of all sightings.

standard deviation (SD), first it was calculated: For the present study, a dolphin group was defined

Number of dolphin/km =

as dolphins no more than 500 m apart, within an area Total number of dolphins sighting (1) of similar hydrological characteristics [1]. High

Total distance covered of the river (km) turbidity of the Indus river prevented dolphins being

Mean = ∑ X / n

n-( 2 ∑X/n) (3) sighted, the vessel continued moving downstream but

sighted prior to surfacing. When a dolphin was 2 SD = √ ∑X /

2.5 Identification of Threats to Dolphin Population active surveying for new dolphin groups was

temporarily suspended while observers focused on The tools were used for identification of threats to obtaining an accurate group size estimate. All

dolphin population: use of questionnaire, informal

42 Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan

discussion, discussion with nomads near the catchment (Table 1). Sum of the low and high estimate of group area, interviews, and personal observations.

size were 43 and 64 individuals (Tables 2 and 3), respectively. Encounter rates increased as the survey

3. Results

proceeded downstream to Ramak (Table 1). The best

3.1 Distribution and Abundance group size estimated between Saggu near Meetapur village and D.I. Khan bridge was 04 individuals

A total of 103.5 km of survey effort was conducted, (0.13 dolphins/km), between D.I. Khan bridge to consisted of 30.5 km of the Indus river from Saggu Miran 21 individuals (0.46 dolphins/km) and between near Meetapur village to D.I. Khan bridge, 46 km Miran and Ramak 29 dolphins (1.07 dolphins/km) from D.I. Khan bridge to Miran (near Indus river ), were recorded. A summary of survey results for and 27 km from Miran to Ramak (near Indus river). dolphin population is presented in Table 1. Sighting conditions were generally excellent, with An abundance estimate of 64 individuals was 90% of effort conducted in river surface state 0% or calculated from sum of the highest estimated group 1% and 100% with clear visibility. All potential size. Encounter rates increased as the survey dolphin habitats were surveyed, including main and proceeded downstream to Ramak (Table 2). Sum of secondary channels. the highest group size estimate between Saggu near Dolphins were present in approximately 64.4 km Meetapur village and D.I. Khan Bridge was 05 of the Indus river in three areas, separated from one individuals (0.2 dolphins/km), between D.I. Khan another by disturbance of too many vessels traffic of bridge to Miran 27 individuals (0.6 dolphins/km) and crane and duck hunters. Approximately 53.7% of the between Miran and Ramak 32 individuals dolphin population occurred in 27 km of river length (1.2 dolphins/km) were recorded. A summary of from Miran onward to Ramak, 38.9% occurred in 46 survey results for dolphin population is presented in km from D.I. Khan bridge to Miran and 7.4%

Table 2.

occurred in 30.5 km from Saggu, near Meetapur An abundance estimate of 43 individuals was village to D.I. Khan bridge (Table 1). No dolphin was calculated from sum of the lowest estimate group size. seen near the camp on the Miran Spur in Indus river. Encounter rates increased as the survey proceeded Dolphins were occasionally sighted in secondary downstream to Ramak (Table 3). Sum of the lowest channels; generally, rates were very much lower than group size estimated between Saggu near Meetapur those in the main channel. The highest estimate village and D.I. Khan bridge was 04 individuals approximately 50% of the dolphin population (0.13 dolphins/km), between D.I. Khan bridge to occurred in 27 km of river length from Miran onward Miran, 17 (0.37 dolphins/km) and between Miran and to Ramak in the Indus river, 42.2% occurred in 46 km Ramak 22 individuals (0.81 dolphins/km) were from D.I. Khan Bridge to Miran and 7.8% occurred in recorded. A summary of survey results for dolphin

30.5 km from Saggu, near Meetapur village to D.I. population is presented in Table 3. Khan bridge (Table 2). The lowest estimate

approximately 51.2% of the dolphin population

3.2 Group Size

occurred in 27 km from Miran onwards to Ramak, Group size ranged from 02 to 08 individuals with 39.5% occurred in 46 km from D.I. Khan bridge to

03 and 04 individuals group size most frequently Miran and 9.3% occurred in 30.5 km from Saggu, encountered (Table 4). As dolphin encounter rate and near Meetapur village to D.I. Khan bridge (Table 3). population abundance increased, there was a An abundance estimate of 54 individuals was corresponding increase in mean group size. Between calculated from sum of the best estimated group size

Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan

Table 1 The best estimated group size of Platanista minor in Northern Pakistan, surveyed in March 2009.

Tributaries of Indus river Abundance Distance surveyed Dolphin Mean group size ± SD a km Total population km (min-max) % Saggu-D.I. Khan bridge a 4 30.5 0.13 - a 7.4

D.I.Khan bridge-Miran a 21 46 0.46 3.5 ± 0.5 (3-4)

53.7 Total for Indus river in Northern Pakistan

Miran-Ramak a 29 27 1.07 3.63 ± 1.7 (2-8)

100 a Surveyed area: 103.5 km near Indus river; SD: standard deviation; -: data was not obtained.

Table 2 The highest estimated group size of Platanista minor in Northern Pakistan, surveyed in March 2009.

Tributaries of Indus river Abundance Distance surveyed Dolphin Mean group size ± SD a km Total population km (min-max) % Saggu-D.I.Khan bridge a 5 30.5 0.2 - a 7.8

D.I.Khan bridge-Miran a 27 46 0.6 4.5 ± 0.5 (4-5)

50 Total for Indus river in Northern Pakistan

Miran-Ramak a 32 27 1.2 4 ± 2.06 (2-9)

100 a The same with Table 1.

Table 3 The lowest estimated group size of Platanista minor in Northern Pakistan, surveyed in March 2009.

Section of the Indus river Abundance Distance surveyed Dolphin Mean group size ± SD a km Total population km (min-max) % Saggu-D.I. Khan Bridge a 4 30.5 0.13 - a 9.3

D.I. Khan Bridge-Miran a 17 46 0.37 2.83 ± 0.69 (2-4) 39.5 Miran-Ramak a 22 27 0.81 2.75 ± 2.11 (1-8) 51.2

Total for Indus River in Northern Pakistan

100 a The same with Table 1.

Table 4 Location and group size of all dolphin sightings.

1). The largest dolphin group, which comprised 08 Position Group Size

Date individuals, was located far away from the camp

Latitude N Longitude E Best High Low (near Miran), immediately downstream of Ramak. Mar. 03, 2009 31.56691

Mar. 04, 2009 31.45165

3 4 2 Between the Miran and Ramak in the Indus river, Mar. 04, 2009 31.44368

4 5 3 dolphin encounter rate was extremely high and Mar. 04, 2009 31.35706

3 5 3 dolphins were distributed almost continuously along Mar. 04, 2009 31.28870

4 4 3 the river channel with no distinct separation between Mar. 04, 2009 31.27654

3 4 2 aggregations. The “groups” were used to facilitate Mar. 05, 2009 31.22164

3 3 2 counting in this river section, but reported group sizes Mar. 05, 2009 31.22540

2 2 1 did not necessarily reflect social organization or Mar. 05, 2009 31.21466

8 9 8 affiliation of this population (Tables 1, 2, 3, and 4). Mar. 05, 2009 31.21159

4 4 3 3.3 The Highest Group Size

Mar. 05, 2009 31.21042

The highest group size ranged from 02 to 09 the D.I. Khan bridge and Miran 3.5 ± 0.5

Mar. 05, 2009 31.20641

individuals with 04 individuals group size was the individuals/group were recorded. The largest mean

most frequently encountered (Table 4). As dolphin group size 3.63 ± 1.7 individuals/group was recorded

encounter rate and population abundance increased, between Miran and Ramak, in the Indus river (Table

there was a corresponding increase in mean group

44 Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan

size. Between D.I.Khan bridge and Miran 4.5 ± 0.5 on rivers to provide them with food, drinking water, individuals/group were recorded and the largest mean

and the means to wash and take care of their livestock. group size 4.0 ± 2.06 individuals/group were recorded

Threats to dolphins include too much vessel-traffic, between Miran and Ramak (Table 2). The largest

disturbance by the crane, duck-hunters, and no dolphin group, which comprised 09 individuals, was

effective-protection. In addition, water is required to located far away from the camp (near Miran)

irrigate crops and to supply industry. Personal immediately downstream of Ramak (Table 4).

observation shows that Indus river and its tributaries are highly polluted with domestic and industrial

3.4 The Lowest Group Size untreated sewage and toxic effluents of medium and

The lowest group size ranged from 01 to 08 heavy industries. Rivers are modified for generating individuals with 02 and 03 individuals group sizes

hydro-electric power, controlling floods, increasing were the most frequently encountered (Table 4). As

navigation access, and for irrigation, domestic, and dolphin encounter rate and population abundance

industrial use, and for the locations of major water increased, there was a corresponding increase in

developments. All of these activities result in the mean group size. Between D.I. Khan bridge and

degradation and loss of aquatic habitat of dolphins. Miran 2.83 ± 0.69 individuals/group were recorded

Accidental trapping and nature of dolphin mortality and the largest mean group size 2.75 ± 2.11

from entanglement in gillnets has not been individuals/group were recorded between Miran and

systematically researched in any area. Threats vary Ramak in the Indus river (Table 3). The largest

geographically in their importance, but generally dolphin group, which comprised 08 individuals group

include accidental killing during fishing-operations, size, was located in the Indus river, very far away

habitat-loss and population-fragmentation from water from the camp (near Miran) immediately downstream

development. However, it has been recognized that of Ramak. Location and group size all dolphin

the threats have increased as the use of nylon gill nets sightings are presented in Table 4.

has increased.

3.5 Threats to Indus River Dolphin

4. Discussion

Hunting or poaching of dolphins within the

The population density of P. minor wa s declined

Northern Pakistan is not known completely. Dolphins from approximately 3400 km of the Indus main stem are particularly vulnerable to the high levels of

and its tributaries in the 1870s [4] to approximately anthropogenic activities because of their restricted

1000 km of only the main stem today. This decline habitat. During present research, discussion with the

was primarily due to fragmentation of the dolphin nomads near the catchment area of Indus river shows

population by irrigation barrages combined with that their forefathers caught the dolphins, extracted

habitat degradation caused by large-scale water oil and used it for cooking. Some nomads eat the

abstraction from the Indus river system [26] . At

dolphin meat even now. Interviews with communities present, in the field, measures were taken to increase near the catchment area show that Indus river

detection opportunities by using experienced dolphins are particularly vulnerable to the activities

observers, non-motorized vessels traveling at a of humans because of the restricted confines of their

relatively slow mean survey speed, a rear facing habitat. Ever-increasing human populations, often

observer to detect dolphins missed by the forward living in impoverished conditions, make increasing

facing observer team and by surveying only during demands of water and riparian resources. People rely

excellent or good viewing conditions.

Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan

A total of five subpopulations of the Indus river dolphin were found, the largest estimated as at least 725 individuals and the smallest as two [1]. As population size decreases, the risk of extinction increases due to the effects of inbreeding, loss of genetic diversity and the allee effect, as well as demographic and environmental stochastic events [27, 28]. Due to the influence of these processes, it is probable that the smallest subpopulations, Jinnah–Chashma (02 individuals) and Sukkur-Kotri (18 individuals) are too small to persist in the long-term. In addition, a third subpopulation, between Chashma and Taunsa Barrages (84 individuals) is extremely vulnerable. These three subpopulations would likely qualify for listing as “critically endangered” by the International Union for Conservation of Nature (IUCN) due to their small population sizes [1]. Further efforts need to be made to support and expand their conservation work. Toosy et al. [3] recorded by first radio transmitter tagging that the dolphin was able to cross the Sukkur barrage during

the low water flow at 14,000 m 3 of water in second,

moving both ways downstream and upstream. In future, the possibility of fixing a Satellite GPS transmitter will

be explored. In 1972, dolphins were protected under the Wildlife Act of Sindh and in 1974 the government of Sindh declared the Indus river between the Sukkur and Guddu barrages as a dolphin reserve. The government of Punjab prohibited deliberate killing of dolphins, though the Punjab Wildlife Protection Act, 1974 and established the Taunsa Wildlife Sanctuary and Chashma Wildlife Sanctuary in 1983 and 1984, respectively [26, 29, 30]. Sindh Wildlife Management Board developed research project in

collaboration with Worldwide Fund for Nature (International) and Volkart Foundation from 1977 to 1980. Under that project, some biological and behavioral studies were carried out and positive signs were recorded in its reproductive system [16]. In the present research, which was conducted in Northern Pakistan, the highest group size sighted was 09

individual, therefore, the catchment areas, Saggu, D.I. Khan bridge, Miran and Ramak must be declared as dolphin reserves (Table 5).

Several things have actually worked in favor of the Indus river dolphin’s survival. First, authorities within the Sindh, Wildlife Department and Sindh Wildlife Management Board acted rapidly and effectively in response to Pilleri et al.s’ alarm of the early 1970s. However, draconian were taken the steps to stop dolphin hunting probably saved the species from extinction in Sindh. The vociferous nongovernmental organizations have been taken interest in conserving the Indus river dolphin, specifically World Wide Fund for Nature, Pakistan and IUCN play a vital role in supporting the work of government agencies and in trying to raise awareness and educate the public. Second, the implications for dolphin conservation are actually not simple. As Reeves et al. [30] pointed out that no thought was given to the consequences for dolphins when the existing dams and barrages were constructed. The dolphins’ lack of economic importance has made it easy for development planners and government officials to ignore. It could

be argued that giving the animals some tangible value, for example as lures to attract tourism, would benefit conservation. Third, there is almost no motorized vessel traffic in the Indus. As underwater noise caused

Table 5 Proposed dolphin protected areas, “dolphin reserves” in Northern Pakistan based on the present survey in March 2009.

River River stretch

Length (km) Selection criteria

Indus river

Saggu-D.I.Khan bridge a 30.5 Low dolphin abundance and low dolphin per km D.I.Khan bridge-Miran a 46 Medium dolphin abundance and potential for future dolphin tourism Miran-Ramak a 27 High dolphin abundance and high group size

a The same with Table 1.

46 Biological Status of Indus River Dolphin (Platanista Minor Owen) in Indus River, Northern Pakistan

by boat engines is a problem for river dolphins [31, lures to attract eco-tourism, would be benefited to 32]. These works of enormous conservation is

conservation of the Indus river dolphin. potentially significance and will depend on what

Incidentally caught dolphins should be released in fraction of the dolphin catch is deliberate, and of course

reasonable health if the fisherman did not have on whether in fact some incidentally caught dolphins

incentive to kill them.

could be released in reasonable health if the fisherman Government should be protected proposed dolphin did not have incentive to kill them for their purpose.

protected areas, “dolphin reserves” in Northern Pakistan These findings will however, need to be communicat-

based on the present survey March 2009 (Table 5). ing to a broad audience of fishermen in the region. It is

If appropriate steps for conservation are not suggested that the population be studied throughout

immediately taken, then the Indus river dolphin is the year at least where they are in fair numbers.

likely to soon become extinct from Indus river. Similarly, the people of the area should be made