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THE INTERACTION EFFECT OF PHYTOHORMONES AND TRACE ELEMENTS ON THE PHYSIO
MORPHOLOGICAL CHARTERISTICS OF CERTAIN PLANTS
• % . ^ : 11
f/^ if. DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
•' iWasfter of ^l)ilo0opl)p IN
%\ ry-x B O T A N Y
^/f
BY
N A H E E D A K H T A R
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2003
2 9 St? 2004
DS3417
Dr. AQIL AHMAD M.Phil,,Ph.D.
PD.Res. Train (Denmark)
PLANT PHYSIOLOGY SECTION DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH-202002, U.P. (INDIA)
Phone : 91-571-408009 FAX : 91-571-702016 E-mail : aqil_ahmad(^lycos.com
Date :
CERTIFICATE
This is to certify that Ms. Naheed Akhtar has worked under my
supervision for the M.Phil, degree in Botany. She has fulfilled all conditions
required to supplicate the M.Phil, degree. I, therefore, approve that she may
submit her dissertation entitled "The interaction effect of phytohormones
and trace element on physio-morphological characteristics of certain
plants".
(Dr. Supervise
ACKNOWLEDGEMENT
In the name of God, the most beneficient and most merciful.
I am sincerely thankful to my esteemed supervisor, Dr. Aqil Ahmad,
Reader, Department of Botany, for his sagacious and skilful guidance.
I am heartly grateful to Prof. Samiullah, Chairman, Department of
Botany, for providing me the necessary facilities that I needed to carry out
this work.
I express my indebtness to Dr. Arif Inam, Dr. Nafees A. Khan, Dr.
Firoz Mohammad, Dr. M.M.A. Khan.
I am thankful to Dr. Shamsul Hayat and Dr. Qazi Fariduddin for their
helpful suggestions.
I am grateful to K.C. Engvild for the geneioojsupply of samples of
chloroindole auxins (4 Cl-IAA).
I owe an expression of thanks to my seniors Mr. Irfan Ahmad, Miss
Shahla Saeed, Lalita Gupta and profuse thanks to my other lab mates Saba Azad,
Dilshada Tabbassum. This research has been supproted by my loving parent's,
my sister's Hoor, Qamar, my Brother W.U. Khan DuiTani and Ehran Khan, and
Insia.
Finally thanks to all my near and dear ones.
(NAHEED AKHTAR)
CONTENTS
S.No. Page No.
1. Chapter 1 : Introduction 1 - 2
2. Chapter 2 : Review of Literature 3 - 6
3. Chapter 3 : Materials and Methods 7 - 12
4. Chapter 4 : Results 13 - 22
5. Chapter 5 : Discussion 23 - 26
6. Bibliography 27 - 37
7. Appendix 38 - 40
QUofite^ - i
J iiZ^aotuctian^
CHAPTER-!
INTRODUCTION
Mature seeds are characteristically loaded with a sufficient amount of
reserves (i.e. proteins, carbohydrates, organic phosphate and various other
inorganic substances/molecules). They support the process of seed
geiTnination and the grovslh of the young seedling, by the time an autotrophic
plant is established.
The seeds on hydration take up a large quantity of water and leak a
certain quantity of sugars, organic acids, phenols, inorganic phosphates and
potassium. These hydrated seeds use a lai'ge volume of oxygen to activate and
hydrate the mitochondrial enzymes, involved in ki'ebs cycle and electon
ti-ansport chain. Numerous enzymes are either released from the inactive
foims or synthesized a fresh (Bewley and Black, 1985; Bernhardt et al.,
1993). The breakdown of complex resei've substances to the simpler forais
and their ti'ansport fi'om endospeinVcotyledons to the embryonic axis for
being utilized as respiratory substrate, building block or regulator of
metabolism (Davies and Slack, 1981).
The phytohoimones play important role in the regulation of seed
geraiination. However, it is generally agreed that indole-3- acetic acid (lAA)
is the major and most abundant auxin in plants. lAA plays a crucial role in the
regulation of plant growth and development (Aiteca, 1997). The occuiTence
of their hallogenated forms (chloro substituted) is rare in plants. 4 Cl-IAA has
been identified in the exti'acts of Vicieae (Gandai* and Nitsch, 1967; Marumo
el al., 1968), immatm-e seeds, the shoot, root and the cotyledons of 3-d old,
etiolated seedlings of Pisiim (Schneider et al, 1985). Similaiiy, it is also
found in Pinus sylvestris (Emstsen and Sandberg, 1986).
Chlorosubstituted auxins have been tested in various bioassays and
are reported to stimulate the rooting and ethylene production in leafy cuttings
(Ahmad et al., 1987), the synthesis of specific enzymes in detached
cotyledons (a-amylase; Hirasawa, 1989) and pea seedlings (NR activity;
Ahmad and Hayat, 1999), imbibed wheat grains (a-amylase; Ahmad et a l ,
2001a) and the plants of mustard (nitrate reductase and carbonic anhydiase
activities; Ahmad et al., 2001b). Nickel (Ni) has been reported as a growth
stimulant as well as a retai'dant (Mishi-a and Kai; 1974). In some cases, Ni
stimulated the growth of the plants, at low concenti-ations (Smith, 1943;
Dobrolyubskii and Slawo, 1957), whereas at higher concenti'ations, it had
definite growth retai'ding effects (Kumar and Bisht, 1986; Singh, 1986). There
ai e reports that excess of nickel depresses catalase activity (Dekock et al.,
1960; Agai-wala and Kumar, 1960; 1962), whereas peroxidase activity
increased (Kariev, 1969; Vlasyuk and Galinskaya, 1970). It has also been
reported that application of nickel to the soil, the culture solution or as a foliar
spray decreases chlorophyll content (Nicholas, 1952; Mishi-a and Kar, 1974).
Keeping in view the important regulatory functions of chloroindole
auxins and the effect of nickel on the metabolism, the seed gennination was
used as a marker to test the possibility of overcoming the ill effect of higher
concenti-ation of nickel by giving a pre-sowing seed ti-eatment with dilute
solutions of 4 CI lAA. As this auxin is many times more active than lAA and
is of natural occuiTence (Reinecke et al., 1995).
QUofUe^ - 3.
KeiAl e{ue44jL a
JL ite^^G^t44^e^
CHAPTER-2
REVIEW OF LITERATURE
Geraiination of the seeds is an expression of metabolic activities
initiated by its hydration and regulated by a number of factors homed in and
outside the seeds. One of these factors is the impact of the hormones,
contiibuted by mother body and stored in the seeds, during its maturation
(Aiteca, 1997).
Indole-3-acetic acid (lAA) is of universal occuiTence in the plants
(Davies, 1995). However, their chlorosubstituted forms are rai-ely found in
plants. Monochlorindole acetic acid (4 Cl-IAA) has been exti'acted fi-om
Viciae and its methyl ester from the inmiature seeds and the other plant parts
of Pisimi (Gandar and Nitsch, 1967; Maiiimo et al., 1968; Schneider et al.,
1985). The presence of auxins in the medium favour the germination of seeds
of tomato, Sinapis alba, Dolichos bijloras, Machilus edulis, Citrus reticulata,
Allium cepa, Michelia and Tamarandus indica (Sheteawi, 1993; Fargasova,
1994; Nanda and Nanda, 1995; Sundriyal and Sundriyal, 2001; Kalita and
Singh, 2002; Kanaujia.e/ al, 2002; Yan et al, 2002;.Vanangamudi, 2003).
Similarly, 4 Cl-IAA also stimulated the gennination of wheat seeds (Ahmad
et al, 2001a) and the elongation of the coleoptile of the seedlings of maize
(Waldemar and Burdach, 2002).
The enzyme, nitrate reductase (NR), responsible for the initiation of
the process of nitiate reduction is nonnally induced to be sjmthesized by the
substi-ate (Hewitt and Afridi, 1959) but the exogenous application of the
hoiTnones (GA, cytokinin and auxin) also enhance its level in various plant
organs (Hanischtencate and Breteler, 1982; Stanko, 1993; Ahmad, 1994;
Alimad ct a/., 2001b). Moreover, Ahmad (1988) noted the auxins do not have
any impact on the niti-ate content but elevated the level of NR in the leafy
cuttings of pea. Moreover, among the chloro-substituted auxins, 4 Cl-IAA had
a maximum impact on NR activity in various organs of the seedlings of Pisiim
sativum (Alunad and Hayat, 1999).
Seed, total protein content or its two fraction (soluble and insoluble)
increased, on being treated with GA, lAA, IBA, ABA or cytokinin alone or in
combination with each other, dming germination in sunflower. Magnolia
denudala, Limim iisitatissimum, Vigna radiata, maize, Hordeiim vulgare,
Pinus radiata, Albizia lebback (Goswami and Shiivastava, 1988; Yang et al.,
1991; Dua et a}., 1991; Totawat and Saxena, 1992; Dogra and Thukral, 1994;
Pom-, 1997; Li et al, 2001; Ilango, 2003). Similarly, these hormones fed at
the time of geimination of the seeds oiLinum iisitatissimum, chickpea, Vicia
fata, Tamarandus indica, wheat and sorghum enhanced their carbohydrate
contents (Dua and.Mahajan, 1991; Gaber, 2000; Kam- and Gupta, 2000; Radi,
2001; Ilango, 2002; Bhatia, 2002).
Brown et al. (1987) fust esatablished the necessity of nickel in the
seed geiinination of barley. Moreover, nickel deficiency resulted in poor
growth of the seedlings, chlorosis and necrotic lesions in the leaves (Dalton et
al, 1988). The physiological level of Ni "" promoted seed germination in
Pisuni sativum (Singh, 1986). However, a concenti-ation above that decreased
the gennination rate in groundnut, maize, chickpea, rice, alfa-alfa, Citnis
reticulata and cabbage (Leena and Ramanujam, 1992; Hiullier, 1996; Khan et
al., 1996; Wang et al., 2000; Peralta et al., 2001; Hasan 2002; Panday and
Sharma, 2002). The exact natoi-e of Ni'"'' mvolvement in the seed gennination
is not clear. However, it is known to be the basic component of the enzymes.
The seeds of Vicia faba and wheat treated with lAA and GA3 and
grown under different salinity, proline content increased with increasing
salinity, while lAA and GA3 ameliorated the adverse effect on proline content
(Aldesuquy, 1998; Shukry and Bassiouny, 2002; Gherroucha et ai, 2003).
Whereas, some heavy metals such as nickel, cadmium and lead enhanced
accumulation of proline in cabbage leaves and cucumber seedling (Talanova
et ai, 1999; Pandey and Shaima, 2002).
Qhafite>fi - 3
Mate^lcU^ a4t d
MetUach
MATERIALS AND METHODS
S.No. Page No.
3.1 Proposed study 7
3.2 Seeds 7
3.3 Experiment 7
3.4 Per cent germination 8
3.5 Rate of water uptake 8
3.6 Relative water content 9
3.7 Estimation of nitrate reductase activity 9
3.7.1 Procedure 9
3.7.2 Colour development 9
3.8 Estimation of protein 10
3.8.1 Extraction 10
3.8.2 Colour development 10
3.9 Estimation of proline 11
3.10 Estimation of carbohydrate 11
3.10.1 Colour development 12
3.11 Statistical analysis 12
CHAPTER-3
MATERIAL AND METHODS
3.1 Proposed study
To achieve the objectives &amed in chapter one, following studies
were conducted to explore the response of chickpea seeds (Cicer arietimim L.
cv. Avarodhi) to 4 chloro-indole-3-acetic acid (4 Cl-IAA) and or nickel
(Ni" " ), during the early stage of imbibition, under conti'oUed conditions.
3.2 Seeds
The authentic seeds were obtained from National Seed Coiporation
Ltd., New Delhi. The healthy seeds were surface sterilized with 0.01% of
mercmic chloride solution, followed by repeated washings with double
distilled water.
3.3 Experi >ment
The experiment was conducted in two phases (A and B) to study the
effect of 4 Cl-IAA and nickel, alone as well as in varied combinations.
(A) The surface sterilized seeds were soaked for 8 hours in double distilled
water (DDW), aqueous solution of 4 Cl-IAA (10'^ or 10" M), or Ni "*"
(50 or 100 or 200 ppm).
(B) The surface sterilized seeds were fnst allowed to imbibe in 50, 100 or
200 ppm of Ni" ^ in sterilized petiiplates for 4 hours and subsequently
tiansfeiTcd, after being soaked with blotting paper, to other petiiplates
containing 10" or 10' M of 4 Cl-IAA, for additional 4 hom-s.
These treated seeds (A and B) were thoroughly washed with DDW to
remove the adhering solution and transferred to sterilized petiiplates.
containing cotton moistened with DDW the seed were allowed to germinate,
in dai k, in a B.O.D. incubator run at 25 ± 2°C. The sampling was done after
24, 48 and 72 hom-s of soaking, that also included the duration of the soaking
ti'eatment.
The following parameters were studied in the germinating seeds :
1. Gennination (%)
2. Rate of water uptake
3. Relative water content
4. Nitfate reductase activity
5. Protein content
6. Carbohydrate content
7. Proline content
3.4 Per cent Germination
It was calculated by employing the following formula :
Number of geiminated seeds Per cent gennination = x 100
Total number of seed
3.5 Rate of Water Uptake
The water taken up by the seeds was calculated by adopting
following foimula:
Water uptake (W) = W2 - Wi
Where, Wi = weight of seeds, before soaking
W2 = weight of seeds, after drying at 60°C
Rate of water uptake = (W1AV2) x 100
3.6 Relative Water Content
The relative water content (R.W.C.) was calculated by employing the
foimula :
Fw - Dw R.W.C.= xlOO
Tw-Dw
Where, Fw = Weight of seeds, before soaking
Tw = Weight of seeds, after soaking
Dw = Diy weight of seed.
3.7 Estimation of nitrate.reductase activity
Niti-ate reductase activity (NRA) in the seeds was estimated
according to the method of Jaworski (1971).
3.7.1 Procedure
At each sampling, the seeds were cut into very thin slices with the
help of razor. 200 mg of slices were weighed and transfeixed to plastic vial
(25 cm") containing 2.5 ml of phosphate buffer (Appendix 1.1), 0.5 ml of
0.2M potassium nitrate solution (Appendix 1.2) and 2.5 ml of 5%
isopropanol. At last two drops of chloramphenicol were also added to check
the bacterial growth in the sample. These vials were incubated in the daik in a
B.O.D; incubator, iim at 27±2°C, for 2 hom's.
3.7.2 Colour development
To the test tube, 0.4 ml of test extract and 0.3 ml each of
sulphanilamide (Appendix 1.3) and naphthyl ethylene diamine
dihydrochloride (NED-HCl) were added (Appendix 1.4), pink colour
appeared. The samples were left for 20 minutes for maximum colom-
development. It was diluted to 5 ml with DDW and each sample was read at
540 nm, using specti-ophotometer.
10
A standard curve was plotted using graded concentrations of sodium
nitiite. Optical density of the test extract was compared with that of the
standai'd cui-ve. NRA was calculated in terais of n mol NO2 g' h' f w.
3.8 Estimation of protein
Total protein content, in the samples, was determined following the
method of Lowiy et al. (1951).
3.8.1 Extraction
50 mg of oven dried seed powder was ti'ansfen-ed to a mortar. The
sample was grind with the addition of 1 ml of trichloroacetic acid (5%)
(Appendix 2.1). It was tfansfen-ed to a centifuge tube with repeated washings
and final volume was made up to 5 ml with trichloroacetic acid complete
precipitation of the proteins was allowed to take place by leaving the sample
for about 1 horn". The material was centrifiiged at 4000 rpm for 15 minutes
and the supernatant was discarded. 5 ml of sodium hydroxide (IN) (Appendix
2.2) was added to the residue and mixed well by shaking. It was left for 30
minutes in a water bath at 60°C so that the precipitated proteins may be
completely dissolved. After cooling for 15 minutes, the mixtm-e was
centtifuged at 4,000 ipm for about 15 minutes and the supernatant, containing
protein fi-action, was collected in 25 ml volumetric flask, and volume was
made up to the mai'k with IN sodium hydi'oxide and used for the estimation.
3.8.2 Colour development
One ml of sodium hydroxide exti-act was transferred to a test
tube and 5 ml of Reagent D (Appendix 2.3) was added to it. The solution was
mixed well and allowed to stand at room temperature for 15 minutes. 0.5 ml
of Folin-phenol reagent (Appendix 2.4) was added rapidly with immediate
mixing. The blue colour developed. It was left for 30 minutes for maximum
11
colour development. The per cent transmittance of this solution was read at
660 nm, using specti-ophotometer. A blank was simultaneously run with each
sample. The total protein content was calculated by comparing the optical
density of each sample with a calibration cui-ve plotted by taking known
gi-aded dilutions of a standai'd solution of bovine semm albumin.
3.9 Estimation of proline
50 mg of fresh sample was transfeired to a mortar. Sample was grind
with the addition of 10 ml of aqueous sulphosalicylic acid (3%) (Appendix
3.1) and filtered through nitro cellulose filter paper. In a test tube, 2 ml of the
test exti-act and 2 ml each of glacial acetic acid and ninhydrin (Appendix 3.2)
were pipetted. The solution was mixed properly and kept in boiling water bath
at 60°C for 1 hour. To stop reaction, the test tube was placed in ice bath for 10
min. To this, 4 ml of toluene was added and mixed well by shaking for 20-30
seconds, red colour developed. Aspirate the toluene layer. The solution was
kept at room temperature for 30 minutes. Measui'e the absorbance at 520 nm
using specti'ophotometer. A blank was run with each set of samples.
The total proline extent was calculated by comparing the optical
density of each sample with a caliberation curve plotted by taking known
graded dilutions of pure proline.
3.10 Estimation of carbohydrate
50 mg of oven dried seed powder was transferred to a mortai\ Sample
was grind with the addition of 5 ml of ethyl alcohol (80%) (Appendix 4.1). It
was heated on a water bath at 60°C for 10 minutes. Sample was cooled and
centiifiiged at 4,000 rpm for 15 minutes. Supernatant was transferred to 25 ml
volumetiic flask with repeated washings. Volume was made up to maik with
ethyl alcohol (80%). To the residue, 5 ml H2SO4 (1.5N) (Appendix 4. 2) was
12
added and heated on water bath at 90°C, for 2 hrs. Sample was cooled and
centiifuged at 4,000 rpm for 15 minutes. Supernatant was collected in 25 ml
volumetiic flask with repeated washings. Final volume made up to mark with
DDW.
3.10.1 Colour development
1 ml of soluble or insoluble extract was tfansfeixed to a test tube
containing 2 ml DDW, 5 ml H2SO4 and 1 ml distilled phenol (Appendix 4.3).
The solution was mixed well by shaking. The colour of the solution turned
yellowish orange. The test tube was allowed to cool for about 30 minutes. The
per cent transmittance of this solution was read at 490 nm, using
"specti-ophotometer". A blank was simultaneously mn with each set of
samples.
The total carbohydrate content was calculated by comparing the
optical density of each sample with a calibration curve plotted by taking
known graded dilutions of glucose.
3.11 Statistical analysis
The experimental data was analysed following the standaid
procedure explained by Gomez and Gomez (1984). The 'F' test was applied
to assess the significance of the data at 0.05 level of probability. The enor due
to replication was also determined. Critical difference (CD.) was calculated
to compare the effect of various components by putting the values in the
following foimula :
Standard En'or x 2 CD= / • x t (value) at 0.05
Replicates
QUofUe^ - Jf
o^4xe^4fie4iial
/<e<i^/^<i
RESULTS
S.No. Page No.
4.1 Per cent germination 13
4.2 Rate of water uptake 13
4.3 Relative water content 13
4.4 Nitrate reductase activity (NRA) 14
4.5 Protein content 14
4.6 Proline content 15
4.7 Carbohydrate content 15
CHAPTER-4
RESULTS
4.1 Per cent germination
The emergence of the radicle, out of the seed coat, was used as the
"cale for expressing the geraiination. It was favoured by the seed treatment
vith 4 Cl-IAA. However, both the concentrations (10" and lO' M) had a
iompaiable effect which was about 70% more than the water soaked control
Table 1). Chloroindole auxin partially overcome the inhibitory effect of the
ligher concenti'ation (100/200 ppm) of Ni"^ on the germination. Moreover,
)re-sowing seed treatment with lower concentration of Ni"" slightly favoured
he geimination percentage.
1.2 Rate of water uptake
Its value increased as the seed germination advanced from 24 to 72
lOurs (Table 2). Maximum values were recorded, through out the study, in the
seeds pre-tieated with 4 Cl-IAA. Among the concentrations, of the auxins
ised, higher concentration (10"^M) was more effective than the lower
:oncent-ation (lO'^M). Nickel alone was most inhibitory on water uptake than
in association with 4C1-IAA. Treating the seeds with higher concenti'ations
(100/200 ppm) of Ni^* reduced the values significantly lower than the
contiol.
4.3 Relative water content
The advancement of germination had a linear relation with the
relative water content (Table 3). Pre-sowing seed treatment with either of the
concentiations (10"VlO" M) of 4 Cl-IAA had a significant impact on the
relative water content of the seeds but the foiTner was more effective than the
14
latter, the values were, however, at par with each other. The treatment with
Ni" " , in particular the higher, concentrations (100/200 ppm), decreased the
values significantly but could partially be restored if the treatment with t^i"*'
was fallowed with 4 Cl-IAA.
4.4 Nitrate reductase activity (NRA)
There is a consistent increase in the activity of niti ate reducatse with
the advancement of gennination (Table 4). The level of the enzyme increased
significantly in the seeds, pre-treated with 4C1-IAA. The overall increase by
the auxin, over the control, was more than 25%. However, the lower
concentiatioa (50 ppm) of Ni'"'" made no significant difference on the level of
the enzyme but an increase in the cation above that made a negative impact on
the activity of NR which was significantly lower than the control. This ill
effect of the higher concentrations (100/200 ppm) of Ni" "" could be partially
overcome by the subsequent treatment of the seeds with 4 Cl-IAA. Moreover,
the higher concentfation of the auxin (10"^M) not only overcome the impact of
100 ppm of Ni ^ but the values were significantly more than those of the
conti'ol.
4.5 Protein content
The total protein content increased as the germination advanced
(Table 5). The seed ti'eatment with either of the concentrations of 4 Cl-IAA
elevated the level of total protein content where higher concentration excelled,
over the lower concentration and the values were about 16.2, 24.5 and 19.1%
more than the control. A propoitionate relationship seems to exist between the
concentration of Ni" " , in the treatment solution, and the degree of inhibition in
protein values. However, a partial restoration is possible, depending on the
concenti-ation of the Ni"*"" in the ti'eatment solution. Either of the
15
concentiations of the 4 Cl-IAA could be followed in treatment pattern up to
100 ppm of Ni^* but above that only higher concentration of the auxin
(10"^M)couldbeof some benefit in over coming the adverse effect of Ni .
4.6 Proline content
With each successive stage of germination, the proline content
increased (Table-6) and in a more systematic way in the seeds pre-treated
with Ni"* " . The level of the proline exhibited a linear relationship with the
concentiation of the Ki'* in the treatment solution. However, the values
decreased if the follow up treatment with 4 Cl-IAA was maintained with
either of its concenti^ations. Moreover, the seed ti eatment with 4 Cl-IAA alone
decreased the proline level below that of the conti'ol and to a significant value
by its higher concentration (10"^M).
4.7 Carbohydrate content
A linear decrease in the carbohydi'ate content of the seeds was
obsei-ved with the lapse of time (Table-7). It did not show any change in this
carbohydrate pattern in the seeds treated with 4 Cl-IAA and /or Ni" *.
16
Table 1 - The effect of Nickel (50, 100 or 20 ppm) and /or 4 Cl-IAA on seed gennination (%) oiCicer arietimim L. cv. Avai'odhi, after 72 hours of the imbibition.
Treatment
Control
Nickel (50 ppm)
Nickel (100 ppm)
Nickel (200 ppm)
4C1-IAA(10"^M)
4C1-IAA(10"^M)
Nickel + 4C1-IAA (50 ppm + 10" M)
Nickel + 4C1-IAA (50 ppm + 10" M)
Nickel + 4C1-IAA (100 ppm + 10" M)
Nickel + 4C1-IAA (100 ppm + 10" M)
Nickel + 4C1-IAA (200 ppm + 10" M)
Nickel + 4C1-IAA (200 ppm + 10' M)
CD at 5 %
72 hours
52.6
62.4
42.6
41.2
90.9
92.4
64.2
64.9
56.6
57.2
48.4
49.9
2.6
17
Table 2 - The effect of Nickel (50, 100 or 20 ppm) and /or 4 Cl-IAA on the rate of water uptake (%) by the seeds of Cicer arietinum L. cv. Avai-odhi, after 24, 48 and 72 hours of the imbibition.
Treatment
Contiol
Nickel (50ppm)
Nickel (lOOppm)
Nickel (200ppm)
4C1-IAA(10"^M)
4C1-1AA(10'^M)
Nickel + 4C1-IAA (50ppm+10'^M) .
Nickel + 4C1-IAA (50ppm+10"^M)
Nickel + 4C1-IAA (lOOppm+lQ-^M)
Nickel + 4C1-IAA (lOOppm+lQ-^M).
Nickel + 4C1-IAA (200ppm+ 10" M)
Nickel + 4C1-IAA (200ppm + 10" M)
CD at 5 %
24
0.181
0.211
0.150
0.149
0.263
0.272
0.228
0.231
0.196
0.199
0.178
0.182
0.023
48
0.265
0.298
0.219
0.202
0.387
0.396
0.321
0.330
0.278
0.281
0.248 .
0.250
0.029
72
0.416
0.482
0.332
0.300
0.671
0.701
0.517
0.529
0.456
0.443
0.386
0.390
0.22
18
Table 3 - The effect of Nickel (50, 100 or 20 ppm) and /or 4 Cl-IAA on the relative water content (%) in the seeds of Cicer arietimim L. cv. Avarodhi, after 24, 48 and 72 hours of the imbibition.
Treatment
Control
Nickel (50ppm)
Nickel (lOOppm)
Nickel (200ppm)
4C1-IAA(10-^M)
4C1-IAA(10"^M)
Nickel + 4C1-IAA (50ppm+10"^M)
Nickel + 4C1-IAA (50ppm+10-^M)
Nickel + 4C1-IAA (lOOppm+lQ-^M)
Nickel + 4C1-IAA (100ppm+ 10- M)
Nickel + 4C1-IAA (200ppm+10"^M)
Nickel + 4C1-IAA (200ppm+ 10" M)
CD at 5 %
24
38.63
43.91
32.06
31.14
53.99
55.12
46.41
46.95
40.72
41.21
36.58
36.92
2.1
48
46.29
53.12
38.12
36.86
69.84
70.63
55.93
56.89
49.87
50.19
42.98
43.34
2.7
72
53.23
63.50
42.99
40.61
89.99
92.35
67.14
67.02
57.92
58.06
47.92
48.23
5.2
19
Table 4 - The effect of Nickel (50, 100 or 20 ppm) and /or 4 Cl-IAA on the nitrate reductase activity (n mol N02 g" h" ) in the seeds of Cicer arietmwn L. cv. AVarodhi, after 24, 48 and 72 hours of the imbibition.
Treatment
Contiol
Nickel (50ppm)
Nickel (lOOppm)
Nickel (200ppm)
4C1-IAA(10'^M)
4C1-IAA(10''^M)
Nickel + 4CI-IAA (50ppm+lO'^M)
Nickel + 4C1-IAA (50ppm+10'^M)
Nickel + 4C1-IAA (lOOppm+lO'^M)
Nickel + 4C1-IAA (100ppm+10"^M)
Nickel + 4C1-IAA (200ppm+10"^M)
Nickel + 4C1-IAA (200ppm+10"^M)
CD at 5 %
24
285.26
302.10
249.14
241.38
361.25
354.11
319.94
320.13
289.44
294.42
267.84
270.16
22.03
48
343.13
368.41
282.12
265.28
440.98
445.58
393.52
399.96
372.48
377.98
302.48
321.12
24.92
72
392.42
420.24
326.12
310.22
498.29
504.12
449.36
453.16
416.28
423.36
360.44
365.28
26.88
20
Table 5 - The effect of Nickel (50, 100 or 20 ppm) and /or 4 Cl-IAA on the protein content (%) in the seeds of Cicer arietinum L. cv. Avarodhi, after 24, 48 and 72 houi's of the imbibition.
Tratement
Control
Nickel (50ppm)
Nickel (lOOppm)
Nickel (200ppm)
4C1-IAA(10-^M)
4C1-IAA(10-'^M)
Nickel + 4C1-IAA (50ppm + 10" M)
Nickel + 4C1-IAA (50ppm+10"^M)
Nickel + 4C1-IAA (lOOppm+lQ-^M)
Nickel + 4C1-IAA (100ppm+10"^M)
Nickel + 4C1-IAA (2Q0ppm+10"^M)
Nickel + 4C1-IAA (200ppm+ 10" M)
CD at 5 %
24
19.78
20.44
17.48
16.76
22.99
23.19
22.58
22.63
20,86
21.04
18.59
18.67
1.721
48
22.46
23.73
19.15
18.53
27.96
27.58
25.91
26.32
23.92
24.14 .
20.40
20.94
1.726
72 .
24.03
24.62
20.26
20.02
28.63
28.92
27.60
27.53
25.48
25.52
22.47
22.58
0.81
21
Table 6 - The effect of Nickel (50, 100 or 20 ppm) and /or 4 Cl-IAA on the proline content (fig/g fresh tissue) in the seeds of Cicer arietimim L. cv. Avarodhi, after 24, 48 and 72 hours of the imbibition.
Treatment
Control
Nickel (50ppm)
Nickel (lOOppm)
Nickel (200ppm)
4C1-IAA(10"^M)
4C1-IAA(10-^M)
Nickel + 4C1-1AA (50ppm+10"^M)
Nickel + 4C1-IAA (50ppm+ 10- M)
Nickel + 4C1-IAA (100ppm+ 10" M)
Nickel + 4C1-IAA (lOOppm+lO'^M)
Nickel + 4C1-IAA (200ppm + 10" M)
Nickel + 4C1-IAA (200ppm+ 10" M)
CD at 5 %
24
6.49
7.54
8.86
9.12
6.09
6.12
6.73
6.65
7.33
7.28
7.38
7.43
0.44
48
6.87
8.08
9.52
9.98
6.38
6.43
7.42
7.24
7.82
7.79
7.84
7.98
0.43
72
7.86
9.28
10.99
11.58
7.09
6.44
8.72
8.38
9.02
8.96
8.99
9.17
0.59
22
Table 7 - The effect of Nickel (50, 100 or 20 ppm) and /or 4 Cl-IAA on the carbohydi"ate content (%) m the seeds of Cicer arietinum L. cv. Avarodhi, after 24, 48 and 72 hours of the imbibition.
Treatment
Contiol
Nickel (50ppm)
Nickel (lOOppm)
Nickel (200ppm)
4C1-IAA(10'^M)
4C1-IAA(10'^M)
Nickel + 4C1-IAA (50ppm+lO'^M)
Nickel + 4C1-IAA (50ppm+ 10' M)
Nickel + 4C1-IAA (lOOppm+lQ-^M)
Nickel + 4C1-IAA (100ppm+10"'^M)
Nickel + 4C1-IAA (200ppm+10-^M)
Nickel + 4C1-IAA (200ppm+10-^M)
CD at 5 %
24
62.52
64.67
64.29
63.36
67.09
67.78
66.43
66.67
65.93
66.09
64.69
65.15
5.65
48
60.43
62.39
58.76
58.61
62.46
63.98
62.19
62.03
61.81
61.96
57.84
58.43
5.35
72
58.47
60.03
57.93
57.50
61.63
61.49
60.76
61.17
• 57.64
60.67
57.43
57.56
5.81
GUofiteA. - 5
Jb 0,044^6^0*4^
CHAPTER-5
DISCUSSION
The selected gi-oups of hydi'olytic carbohydi-ates and proteins, located
in the seed coat, attract dipolar water molecules and form a hydrated shell
aiomid them, resulting in the swelling of the seed coat. This favours the enty
of more and more water molecules. The observed pattern of water uptake
which has increased with the progress of the germination (Table 1,2) is,
therefore, an expression if the above fact. A prominence may be obsei-ved in
this phenomenon of the seeds were pre-treated with aqueous solution of a
moderate concentration (10" to lO' M) of 4-chloroindole-3-acetic acid (4C1-
lAA). This is possibly the result of the facilitated diffusion of water molecules
generated by the auxin, at the level of seed coat. However, the effect of heavy
metals on plant water-relations is quite complex and depends on the metal
type, its concentration, genotype and the exposure time (Singh and Tewari,
2003). In the present obsei vation (Table 3) relative water content and rate of
water uptake decreased significantly in the seeds given pre-sowing ti'eatment
with the higher concentrations of Ni^*. The seedlings of Brassica juncea were
also reported to give a comparable response to Ni" ^ (Singh and Tewari, 2003).
A shift in the metabolic state of the seeds is simultaneously induced
because of the hydration of the tissues. These sequence of events, initiate
from the activation of the existing proteins and/or the de novo synthesis of
specific proteins (Bewley and Black, 1985). One such process is the induced
synthesis of the enzyme nifrate reductase (NR) whose activity is noted to be
higher with each successive stage of germination (Table 4). Similar
obsei-vations have also been reported by oilieib, (.Tahir and Fai'ooq, 1989; Alvi,
24
et al., 2003; Hayat and Ahmad, 2003). The factors responsible for the
elevation of nifrate reductase aie suggested to be the presence or absence of
nitiate, the substrate (Sai'oop et al, 1998) light (Hayat and Ahmad, 2003) and
the level of phytohormones (Ahmad, 1988; Ahmad and Hayat, 1999 and
Ahmad et al., 2001b). In the present study^ the last factor has been exclusively
studied in association with a cation, nickel (Ni^^). Highly active auxin (4 Cl-
lAA), at moderate concentrations (10" and lO' M), alone or in association
with nickel (50, 100 and 200 ppm) were used to give a pre-sowing seed
tteatment. Table-4 indicates that not only the auxin but the lower
concenti-ation (50 ppm) of the Ni"""" enhanced the level of NR. However, the
higher concentiation (100 and 200 ppm) of Ni""" proved to be inhibitory for
NR activity. Moreover, this ill effect of Ni" , up to a certain concentration,
seems to have been overcome by a foUowup treatment of the seeds with 4 Cl-
lAA (10"^M). It is not sure that how does NR activity is affected by Ni^^ but it
may be suggested that a concenfration above 50 ppm might be getting supra
optimal. However, the synthesis of NR involves the transcription and the
franslation (Jones and Prasad, 1992), the process/s might be getting activated
by the auxin (4 Cl-IAA). Chloroindole auxin has similarly been reported to
have a positive effect of NR in the seedlings of pea (Ahmad and Hayat, 1999)
and those of the mustard (Ahmad et al., 2001b). The other enzjone
significantly affected by 4 Cl-IAA in pea cotyledons (Hirasawa, 1989) and
wheat grains (Ahmad et al., 2001a) is a-amylase. The inhibitory affect of the
higher concentiation of the metal on the activity of NR is in accordance with
the earlier reports (Ramadoss, 1979; Muthuchelian et al, 1988) but no
concrete explanation may be given to the finding. The favourable effect of 4
Cl-IAA on the uptake oi water by the seeds (Table 2) and improved activity
of the enzymes could have Dossiblv been the reasons to activate the nrocess of
25
gennination and the observed gennination percentage, at a particular interval
(Table 1). Ahmad et al. (2001a) and Rani (2003) have also reported
improvement in germination in other crops by 4 Cl-IAA. The response of the
seeds to higher concentrations of Ni * could be a cumulative effect of
reduced water uptake (Table 2), decreased level of enzyme activity (Table 4)
and/or its adverse impact on the stmcture and function of various
biomolecules of the cell as observed for cadmium in Arachis hypogea by
Satakopan and Rajendran (1989).
Plants and their organs are acclaimed to possess more proline on
being exposed to low temperature (Stefi et al, 1978) nutrient stress (Shaima
et al, 1995), heavy metal toxicity (Kastori et al, 1992; Bassi and Sharma,
1993), water sti'ess (Carceller and Fraschina, 1980) and salt stress (Liu and
Zhu, 1997). Nickel had a significant impact on the proline content which
exhibits a significant increase with an increase in the concentration of the
cation (Table 6). Under normal conditions, the synthesis and hydrolysis of the
proteins maintains a definite level of each protein. However, in case of
proline, it has been proposed by Tewari and Singh (1991) that it is the
synthesis of the proline or the hydrolysis which becomes defective, under
certain conditions. Moreover, the prevention of feedback inhibition of the
enz5mies, involved in proline degradation may be a specific reason (Girija et
al., 2002).
Most of the pai'ameters studied exhibited a decrease in their values,
under higher nickel concentrations, dui ing the gennination of the seeds.
Probably Ni"" concentiations developed sti-ess at the level of the cell resulting
in the modification of the cellular metabolism and the prevention of
extension growth (Taiz, 1984). However, a partial recovery was attained by a
26
follow up tieatment with 4 Cl-IAA. It could be an expression of the observed
increase in water uptake (Table 2) favoured by increased membrane
permeability and/or elevation of osmotically active solutes, under the
influence of the auxin (Arteca, 1997). Moreover, the inherent chai-acteristics
of the auxin on cell division and elongation (Arteca, 1997) and transcription
and translation (Reddy,1988) may be additional reasons to explain the
overcoming the ill effect of Ni'''' by 4 Cl-IAA.
CONCLUSIONS
1. The seeds treated with 4 Cl-IAA possessed maximum rate of water
uptake, R.W.C. and per cent germination.
2. The seed treated with 4 Cl-IAA in combination with lower
concentration of Nickel (50 ppm). Significantly have more rate of
water uptake, R.W.C, per cent germination then the seeds treated
with only Ni or water soaked control.
3. Maximum NR activity, protein content was observed in the seeds
pre-treated with 4 Cl-IAA which was closely followed by the
tieatment of 4 Cl-IAA supplemented with lower concentration of Ni
(50 ppm).
4. Seeds treated with higher concentration of nickel (100 or 200 ppm)
inhibited most of the parameters.
5. Nickel treatment increased the proline content while, 4 Cl-IAA
decreased it.
6. Overall, the 4 Cl-IAA may be used to overcome the inhibitoiy effect
of Ni on the process of seed germination.
/^iLliaa^ofiUif
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APPENDIX
PREPARATION OF REAGENTS
The reagents used for biochemical determinations were prepared
according to the following methods :
1.0 Reagents for Estimation of Nitrate Reductase Activity
1.1 Phosphate Buffer (pH 7.5)
(a) 13.6 gm potassium dihydrogen orthophosphate (KH2PO4) was
dissolved in sufficient double distilled water and the final volume
was adjusted to 1 litre.
(b) 17.42 gm. Dipotassium monohydrogen orthophosphate (KH2PO4)
was dissolved in sufficient double distilled water and the final
volume was made to 1 litre.
(c) 160 ml of solution (a) and 840 ml of solution (b) was mixed in
order to get pH 7.5.
1.2 Potassium Nitrate (0.2M)
2.02 gm. Potassium nitrate was dissolved in enough double distilled
water and the final volume was made to 100 ml.
1.3 Sulphanilamide Solution (1%)
1 gm. Sulphanilamide powder was dissolved in 100 ml. 3N HCl (1 ml
HCl + 4 ml water).
1.4 NED-HCl solution (0.02%)
20 mg NED-HCl solution (N-1-nepthyl ethylene diamine
dihydrochloride) was dissolved in double distilled water and final
volume made upto 100 ml.
39
2.0 - Reagent for the Estimation of Protein
2.1 Trichloro acetic acid (5%)
5 ml trichloro acetic acid dissolved in 95 ml DDW.
2.2 Sodium hydroxide solution (IN)
4g NaOH dissolved in sufficient DDW and final volume made up to
100 ml.
2.3 Reagent ' C
Alkaline copper sulphate solution was obtained by mixing 50 ml of
reagent 'A' and 1 ml of reagent 'B'.
2.4 Reagent 'D'
50 ml of 2% sodium carbonate was mixed with 1 ml. of 0.5% copper
sulphate and 1% sodium tartrate (which is mixed in the ratio of 1:1).
2.5 Folin's Phenol Reagent
100 gm sodium tungustate and 25 gm sodium molybdate were
dissolved in 700 ml distilled water in which 50 ml of 85% phosphoric
acid and 100 ml concentrated hydrochloric acid were added. The flask
was connected with a reflux condenser and boiled gently on a heating
mantle for 10 hom"s. At the end of the boiling period, 150 gm. Lithium
sulphate, 50 ml double distilled water and 3-4 drops of liquid bromine
was added to this flask. The reflux condenser was removed and the
solution in the flask was boiled for 15 minutes in order to remove
excess bromine cooled and diluted up to 1 litre. The strength of this
acidic solution (IN) was tested by titrating it with IN sodium
hydroxide using phenolphthalein as an indicator.
3.0 Reagent for Estimation of Proline
3.1 Aqueous Sulphosalicylic Acid (3%)
40
3.2 Acid Ninhydrin Reagent
Dissolved 1.25 gm of ninhydrin in a mixture of warm 30 ml of glacial
acetic acid and 20 ml of 6M phosphoric acid (pH=1.0) with agitation
until it dissolved. Store at 4°C and used within 24 hrs.
4.0 Reagent for Estimation of Carbohydrate
4.1 Sulphuric acid (1.5 N)
10.4 ml sulphuric acid was added to 239.5 ml of distilled water.
4.2 Ethyl alchol (80%)
80 ml ethyl alcohol was added to 20 ml of double distilled water.
4.3 Phenol (5%)
5 ml phenol was added to 95 ml of distilled water.
\ •• -^ I
