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EXPLORING THE POTENTIAL OF MORINGA

(Moringa oleifera) LEAF EXTRACT AS


NATURAL PLANT GROWTH ENHANCER

By

AZRA YASMEEN

DOCTOR OF PHILOSPHY
IN
AGRONOMY

DEPARTMENT OF AGRONOMY
FACULTY OF AGRICULTURE,
UNIVERSITY OF AGRICULTURE,
FAISALABAD, PAKISTAN.

2011

EXPLORING THE POTENTIAL OF MORINGA


(Moringa oleifera) LEAF EXTRACT AS
NATURAL PLANT GROWTH ENHANCER

By

AZRA YASMEEN
M.Sc. (Hons.) Agriculture
96-ag-662
A thesis submitted in partial fulfillment of the requirements for the
degree of

DOCTOR OF PHILOSPHY
IN
AGRONOMY

DEPARTMENT OF AGRONOMY
FACULTY OF AGRICULTURE,
UNIVERSITY OF AGRICULTURE,
FAISALABAD, PAKISTAN.

2011

DEDICATED
TO
MY LOVING AND CARING,
HUSBAND,
MY SWEET SISTERS IN LAW,
MY INNOCENT CHILDREN,
MY RESPECTED MOTHER IN LAW,
AND
MY DEAR PARENTS,
So much I have become is
because of you and
I want to tell you
That I appreciate you thank you
and love you

ACKNOWLEDGEMENTS

Saying of Prophet Muhammad (PBUH) `A person who is not thankful to his benefactor is not
thankful to ALLAH'. All and every kind of praises is upon ALLAH ALMIGHTY, the strength of
universe, who ever helps in darkness & difficulties. All and every kind of respect to His Holy
Prophet Muhammad (PBUH) for unique comprehensive and everlasting source of guidance and
knowledge for humanity.
I would like to extend my heartiest gratitude to Dr. Shahzad Maqsood Ahmad Basra, Professor,
Department of Crop Pysiology, University of Agriculture, Faisalabad, under whose supervision,
scholastic guidance, consulting behavior, this work was planned, executed and completed. I
appreciate and thank to members of my supervisory committee, Dr. Rashid Ahmad, Professor,
Department of Crop Physiology, University of Agriculture, Fasislabad for his helping attitude and
Dr. Abdul Wahid, Professor and Chairman, Department of Botany, University of Agriculture,
Faisalabad, for their valuable and continuous guidelines during the preparation of this manuscript
and kind hearted behavior throughout my doctoral studies. I am also highly indebted to Higher
Education Commission (HEC), Government of Pakistan for granting me Indigenous
fellowship throughout my doctoral study.

Azra Yasmeen

The Controller of Examination,


University of Agriculture,
Faisalabad,

We, the supervisory committee, certify that the contents and form of thesis submitted by
Ms. Azra Yasmeen, Regd. No. 96-ag-662 have been found satisfactory and recommend
that it be processed for evaluation by the External Examiner (s) for award of Degree.

SUPERVISORY COMMITTEE:

CHAIRMAN

: __________________________________
(Dr. Shahzad Maqsood Ahmad Basra)

MEMBER

: __________________________________
(Dr. Rashid Ahmad)

MEMBER

:__________________________________
(Dr. Abdul wahid)

DECLARATION

I hereby declare that contents of the thesis, Exploring the potential of


moringa (Moringa oleifera) leaf extract as natural plant growth enhancer
are product of my own research and no part has been copied from any
published source (except the references, standard mathematical or genetic
models/equations/formulate/protocols etc.). I further declare that this work
has not been submitted for award of any other diploma/degree. The
university may take action if the information provided is found inaccurate at
any stage. (In case of any default, the scholar will be proceeded against as
per HEC plagiarism policy).

Azra Yasmeen
96-ag- 662

ii

TABLE OF CONTENTS

1.

Page
ACKNOWLEDGEMENTS ...
i
DECLARATION.
ii
TABLE OF CONTENTS ..
iii
LIST OF TABLES ....
x
LIST OF FIGURES
xvii
ABBREVIATIONS.. xx
ABSTRACT .
xxii
Introduction .
1

2.

Review of Literature

2.1. Abiotic stresses..


2.1.1. High temperature stress.
2.2.1. Salinity stress
2.3.1. Drought stress
2.2. Mitigation of abiotic stresses
2.2.1. Seed priming
2.2.2 Foliar application ..

7
7
10
15
19
20
20

Materials and Methods ..


3.1. Source of moringa leaves...
3.2. Analysis of moringa leaves...
3.3. Preparation of MLE...
3.4. Plant material.
3.5. Crop season ..
3.6. Experimental sites
3.7. Experiment I: Optimization of moringa oleifera leaf extract (MLE) dilutions..
3.8. Experiment II: Evaluation of MLE as priming agent...
3.9. Experiment III: Evaluating the growth and development of Tomato to
exogenous application of MLE (pot study)...
3.10. Experiment IV: Evaluating the growth and development of pea to
application of MLE (pot study) ....
3.11. Experiment V: Evaluating the response of late sown wheat to foliar
application of MLE under field conditions
3.12. Experiment VI: Mitigating effects of salinity stress in wheat by MLE
application..
3.13. Experiment VII: Mitigating effects of drought stress in wheat by
MLE application .
3.14. Statistical analysis ...

27
27
27
27
30
30
30
30
31

3.

4.

39
42
43
46
48
50

Results and Discussion.. 51


4.1. Experiment 1. Optimization of MLE dilution..

51

4.2. Experiment II: Evaluation of MLE as priming agent


4.3. Experiment III: Evaluating the growth and development of tomato to

57

iii

exogenous application of MLE (pot study). 70


4.4. Experiment IV: Evaluating the growth and development of pea to
exogenous application of MLE (pot study)81
4.5. Experiment V: Response of late sown wheat to foliar application of MLE
under field conditions. 89
4.6. Experiment VI:Response of wheat to exogenous application of MLE
under saline stress conditions 99
4.7. Experiment VII: Response of wheat to exogenous application
of MLE under drought stress conditions .. 113
General Discussion... 121
Summary... 124
LITERATURE CITED.... 126

iv

LIST OF TABLES
Page

Table

3.1.

Bio-chemical composition of Moringa oleifere leaf...

28

3.2

Analysis report for soil filled in pots.....

33

3.3

Dilutions of bovine serum albumin (BSA)...

37

3.4

Preparation of gallic acid standards........

39

3.5

Dilutions for ascorbic acid standard solutions..

41

4.1.1

Mean sum of squares of data for germination index (GI), mean germination
time (MGT), and time to 50% germination (T50) of wheat grown in petri
plates treated with Moringa oleifera leaf extract (MLE).
54

4.1.2

Mean sum of squares of data for final germination percentage, shoot fresh
weight and shoot dry weight of wheat seedling grown in petri plates treated
with MLE
54

4.1.3

Mean sum of squares of data for shoot length, root fresh weight and root dry
weight of wheat seedling grown in petri plates treated with MLE
54

4.1.4

Mean sum of squares of data for root length of wheat seedling grown in
petri plates treated with MLE.........

4.2.1

55

Mean sum of squares of data for emergence index (EI), mean emergence
time (MET), time to 50% emergence (E50) of wheat raised from seeds
primed with different priming agents . 63

4.2.2

Mean sum of squares of data for shoot fresh weight, shoot dry weight and
shoot length of wheat raised from seeds primed with different priming
agents 63

4.2.3

Mean sum of squares of data for root fresh weight, root dry weight and root
length of wheat raised from seeds primed with different priming agents

63

4.2.4

Mean sum of squares of data for leaf area, leaf chlorophyll a and chlorophyll
b contents of wheat raised from seeds primed with different
agents. 64

4.2.5

Mean sum of squares of data for number of grains per spike, 100 grain
weight and grain yield per plant of wheat raised from seeds primed with
different priming agents ...... 64

4.2.6

Mean sum of squares of data for total soluble protein and enzymatic
antioxidant i.e. superoxide dismutase (SOD) and peroxidase (POD) of wheat
raised from seeds primed with different agents
64

4.2.7

Mean sum of squares of data for enzymatic antioxidant catalase (CAT) and
non-enzymatic antioxidants i.e. total phenolic content and ascorbic acid of
wheat raised from seeds primed with different agents
65

4.3.1

Mean sum of squares of data for number of vegetative branches, number of


flowering branches and number of flowers of tomato under exogenous
application of different plant growth enhancers. 75

4.3.2

Mean sum of squares of data for number of fruits, fruit yield plant-1 and leaf
chlorophyll a of tomato under exogenous application of different plant
growth enhancers...
75

4.3.3

Mean sum of squares of data for leaf chlorophyll b, plant height and leaf
total soluble protein of tomato under exogenous application of different
plant growth enhancers.
76

4.3.4

Mean sum of squares of data for super oxide dismutase (SOD), peroxidase
(POD) and catalase (CAT) of tomato leaf under exogenous application of
different plant growth enhancers..
76

4.3.5

Mean sum of squares of data for leaf total phenolic contents (TPC) and fruit
lycopene of tomato under exogenous application of different plant growth
enhancers.
77
4.4.1

Mean sum of squares of data for number of vegetative branches, number of


reproductive branches and number of flowers of pea under exogenous
application of different plant growth enhancers
85

4.4.2

Mean sum of squares of data for number of pods, pods weight plant-1 and
leaf chlorophyll a of pea under exogenous application of different plant
growth enhancers. 85

4.4.3

Mean sum of squares of data for leaf chlorophyll b and total phenolic
contents (TPC) of pea under exogenous application of different plant
growth enhancers..................................................................................... 86

4.5.1

Mean sum of squares of data for LAI (50 DAS) LAI (57 DAS) and LAI (75
DAS) of wheat by exogenous application of MLE under late sown
conditions 93

4.5.2

Mean sum of squares of data for LAI (85 DAS), LAI (95 DAS) and
seasonal leaf area duration of wheat by exogenous application of MLE
under late sown conditions.............................................................................. 93

vi

4.5.3

Mean sum of squares of data for CGR (57 DAS), CGR (75 DAS) and CGR
(85 DAS) of wheat by exogenous application of MLE under late sown
conditions.
93

4.5.4

Mean sum of squares of data for CGR (95 DAS), NAR (57 DAS) and NAR
(75 DAS) of wheat by exogenous application of MLE under late sown
conditions..
94

4.5.5

Mean sum of squares of data for NAR (85 DAS), NAR (95 DAS) and
number of fertile tillers of wheat by exogenous application of MLE under
late sown conditions
94

4.5.6

Mean sum of squares of data for number of grains per spike, 1000 grain
weight and biological yield (m-2) of wheat by exogenous application of
MLE under late sown conditions ..

94

4.5.7

Mean sum of square summaries of the data for grain yield (m-2) and harvest
index (%) of wheat by exogenous application of MLE under late sown
conditions. 95

4.6.1

Mean sum of squares of the data for shoot length, shoot fresh weight and
shoot dry weight of wheat by exogenous application of growth enhancers
under saline conditions
105

4.6.2

Mean sum of squares of data for root length, root fresh weight and root dry
weight of wheat by exogenous application of growth enhancers under saline
conditions .
105

4.6.3

Mean sum of squares of the data for leaf area, leaf chlorophyll a and
chlorophyll b of wheat by exogenous application of growth enhancers under
saline conditions
106

4.6.4

Mean sum of squares of the data for total soluble protein, enzymatic
antioxidants SOD and POD of wheat by exogenous application of growth
enhancers under saline conditions
106

4.6.5

Mean sum of squares of the data for enzymatic antioxidants (Catalase), nonenzymatic antioxidants i.e. total phenolic contents and ascorbic acid of
wheat by exogenous application of growth enhancers under saline
conditions........ 107

4.6.6

Mean sum of squares of the data for Na+, K+ and CI- contents of wheat by
exogenous application of growth enhancers under saline conditions
107

vii

4.6.7

4.7.1

4.7.2

4.7.3

4.7.4

Mean sum of squares of the data for number of grains per spike and 100
grain weight of wheat by exogenous application of growth enhancers under
saline conditions
Mean sum of squares of data for leaf area, leaf chlorophyll a and
chlorophyll b contents of wheat by exogenous application of growth
enhancers under drought conditions.

108

117

Mean sum of squares of data for total soluble proteins, enzymatic


antioxidants i.e. super oxide dismutase (SOD) and peroxidase (POD) of
wheat by exogenous application of growth enhancers under drought
conditions...
117
Mean sum of squares of data for enzymatic antioxidant catalase (CAT), nonenzymatic antioxidant ascorbic acid and total phenolics (TPC) of wheat by
exogenous application of growth enhancers under drought conditions...........
Mean sum of squares of data for leaf K+ contents and grain yield of wheat
by exogenous application of growth enhancers under drought
conditions........................................................................................................

viii

118

118

LIST OF FIGURES
Figure

Page

3.1

Weather data for October 2008 to April 2009.......

31

3.2

Weather data for October 2009 to April 2010.

31

3.3

Standard curve of protein estimation...

37

3.4

Standard curve of Total phenolic content estimation..

39

3.5

Standard curve of ascorbic acid estimation.

41

4.1.1

Effect of different dilutions of Moringa oleifera leaf extract (MLE) on


germination index (a), mean germination time (MGT) (b), time to 50%
germination (T50) (c) and final germination percentage of wheat cv. Sehar2006 under laboratory conditions.
56

4.1.2

Effect of different dilutions of Moringa oleifera leaf extract (MLE) on


shoot fresh and dry weight (a), root fresh and dry weight (b) and shoot, root
length (c) of wheat cv. Sehar-2006 under laboratory conditions
57

4.2.1

Effect of seed priming on emergence index (EI) (a), mean emergence time
(MET) (b) and time to 50% emergence (E50) (c) of wheat cv. Sehar-2006.
(MLE10 =10 times diluted MLE, MLE30= 30 times diluted MLE,
HP=Hydro priming, OFP= On-farm priming)..
66

4.2.2

Effect of seed priming on shoot fresh weight (a), shoot dry weight (b),
shoot length (c), root fresh weight (d), root dry weight (e) and root length
(f) of wheat cv. Sehar-2006. (MLE10= 10 times diluted MLE, MLE30= 30
times diluted MLE, HP=Hydro priming, OFP= On farm priming) 67

4.2.3

Effect of seed priming on leaf area (a), chlorophyll a (b) and chlorophyll b
(c) of wheat cv. Sehar-2006. (MLE10= 10 times diluted MLE, MLE30= 30
times diluted MLE, HP=Hydro priming, OFP= On farm priming)
68

4.2.4

Effect of seed priming on number of grains per spike (a), 100 grain weight
(b) and grain yield per plant (c) of wheat cv. Sehar-2006 (MLE10= 10
times diluted MLE, MLE30= 30 times diluted MLE, HP=Hydro priming,
OFP= On-farm priming) 69

4.2.5

Effect of seed priming on leaf total soluble protein (a), enzymatic


antioxidants (superoxide dismutase, peroxidase and catalase) (b, d, c) and
non enzymatic antioxidants (ascorbic acid and total phenolic contents) (e,
f) of wheat cv. Sehar-2006. (MLE10= 10 times diluted MLE, MLE30= 30
times diluted MLE, HP=Hydro priming, OFP= On farm priming)..
70

4.3.1

Effect of exogenous application of growth enhancer on number of


flowering branches (a) and number of vegetative branches (b) of tomato cv.
Sahil (MLE0, MLE10, MLE20, MLE30 =0, 10, 20, 30 times diluted MLE
respectively, BAP= benzyl amino purine)
78

ix

4.3.2

Effect of exogenous application of growth enhancer on number of flowers


plant-1 (a), number of fruits (b) and fruit weight (c) of tomato cv. Sahil
(MLE0, MLE10, MLE20, MLE30 = 0, 10, 20,30 times diluted MLE
respectively, BAP= benzyl amino purine). 79

4.3.3

Effect of exogenous application of growth enhancer on total soluble protein


(a), SOD (b), total phenolics (c), POD (d), catalase (e) of leaf and fruit
lycopene (f) of tomato cv. Sahil (MLE0, MLE10, MLE20, MLE30 = 0, 10,
20, 30 times diluted MLE respectively, BAP= benzyl amino purine) 80

4.3.4

Effect of exogenous application of growth enhancer on chlorophyll a (a),


chlorophyll b (b) and plant height (c) of tomato cv. Sahil (MLE0, MLE10,
MLE20, MLE30 = 0, 10, 20, 30 times diluted MLE respectively, BAP=
benzyl amino purine)..
81

4.4.1

Effect of exogenous application of different growth enhancers on number


of vegetative branches (a), reproductive branches (b) and number of
flowers per plant (c) of pea cv. Climax. (MLE0, MLE10, MLE20, MLE30
= 0, 10, 20, 30 times diluted MLE respectively, BAP= benzyl amino
purine) ............................ 87

4.4.2

Effect of exogenous application of different growth enhancers on number


of pods (a) and pod yield plant-1 (b) of pea cv. Climax. (MLE0, MLE10,
MLE20, MLE30 = 0, 10, 20, 30 times diluted MLE respectively, BAP=
benzyl amino purine)...................... 88

4.4.3

Effect of exogenous application of different growth enhancers on


chlorophyll a (a), b (b) and total phenolics (c) of pea cv. Climax. (MLE0,
MLE10, MLE20, MLE30 = 0, 10, 20, 30 times diluted MLE respectively,
BAP= benzyl amino purine) ................................................. 89

4.5.1

Effect of exogenous application of MLE on leaf area index (a) and seasonal
leaf area duration (b) of wheat cv. Sehar-2006 under late sown
conditions.................................................................................... 96

4.5.2

Effect of exogenous application of MLE on crop growth rate (a) and net
assimilation rate (b) of wheat cv. Sehar-2006 under late sown
conditions... 97

4.5.3

Effect of exogenous application of MLE on number of fertile tillers (a),


number of grains per spike (b) and 1000 grain weight (c) of wheat cv.
Sehar-2006 under late sown conditions...
98

4.5.4

Effect of exogenous application of MLE on biological yield (a), grain


yield (b) and harvest index of wheat cv. Sehar-2006 under late sown
conditions............................................................................... 99

4.6.1

Effect of exogenous application of different plant growth enhancer on


shoot length (a), shoot fresh weight (b), shoot dry weight (c), root length
(d), root fresh weight (e) and root dry weight (f) of wheat cv. Sehar-2006
under saline conditions..
109

4.6.2

Effect of exogenous application of different plant growth enhancers on leaf


area (a), chlorophyll a (b) and chlorophyll b (c) of wheat cv. Sehar-2006
under saline conditions . 110

4.6.3

Effect of exogenous application of different plant growth enhancers on


total soluble protein (a), SOD (b), ascorbic acid (c), catalase (d), POD (e)
and total phenolics (f) of wheat cv. Sehar-2006 under saline conditions
111

4.6.4

Effect of exogenous application of different plant growth enhancer on leaf


Na+ (a), K+ (b) and Cl- (c) of wheat cv. Sehar-2006 under saline
conditions 112

4.6.5

Effect of exogenous application of different plant growth enhancers on


number of grains (a) and 100 grain weight (b) of wheat cv. Sehar-2006
under saline conditions 113

4.7.1

Effect of exogenous application of different plant growth enhancers on leaf


area (a), chlorophyll a (b) and chlorophyll b (c) of wheat cv. Sehar-2006
under drought conditions. .
119

4.7.2

Effect of exogenous application of different plant growth enhancer on total


soluble protein (a), super oxide dismutase (b), ascorbic acid (c), catalase
(d), peroxidase (e) and total phenolic contents (f) of wheat cv. Sehar-2006
crop under drought conditions.
120

4.7.3

Effect of exogenous application of different plant growth enhancer on leaf


K+ contents (a) and grain yield plant-1 (b) of wheat cv. Sehar-2006 under
drought conditions.. 121

xi

ABBREVIATIONS

Abbreviation

Full

percent

AA

Ascorbic acid

APX

Ascorbate peroxidase

cm

centimeter (s)

CAT

catalase

CGR

Crop growth rate

day (s)

DAS

days after sowing

E50

time to 50% emergence

EI

emergence index

FC

Field capacity

FEP

Final emergence percentage

gram (s)

g m-2

gram per square meter

GR

Glutathione reductase

ha

hectare

ha-1

per hectare

HI

Harvest index

H2O2

Hydrogen peroxide

Potassium

kg

kilogram

kg ha-1

kilogram per hectare

LSD

Least Significant Difference

meter

m-2

per square meter

MDAR

Monodehydroascorbate reductase

MET

mean emergence time

MGT

mean germination time


xii

ml

milliliter

MLE

Moringa leaf extract

mm

millimeter

mM

milli Molar

MPa

Mega Pascal

Nitrogen

NAR

Net assimilation rate

NS

non-significant

O-2

Superoxide radical
-

OH

Hydroxyl radical

Phosphorus

POD

Peroxidase

ROS

Reactive oxygen species

RO

Alkoxy radical

Rs.

Rupees

SLAD

Seasonal leaf area duration

SOD

Superoxide dismutase

T50

time to 50% germination

TPC

total phenolic contents

xiii

Abstract
Among naturally occurring plant growth stimulants, Moringa oleifera has attained enormous
attention because of having cytokinin in addition to other growth enhancing compounds like
ascorbates, phenolics, other antioxidants along with macro- micro nutrients in its leaves. With
these properties, exogenous application of Moringa leaf extract (MLE) was done in wheat, pea
and tomato to evaluate its efficacy as crop growth enhancer The objective was to optimize
dose, method of exogenous application, enhancement of growth, yield and antioxidant level
under normal and abiotic stresses (late sowing, salinity, and drought). The results showed that
30 times diluted MLE priming in wheat under normal conditions was found to be effective.
MLE priming improved the seedling emergence rate, speed and early growth and increased
level of antioxidants, leaf total soluble protein and chlorophyll contents as compared to
MLE10, Hydro priming, On-farm priming and CaCl2 priming. Large number and heavier
grains were obtained in plants raised from MLE30 primed seed which resulted in highest grain
yield per plant. All the priming treatments gave more yield than non primed control. Foliar
spray of MLE caused an increase of 10.73, 6.00, 10.70 and 4.00% in 1000 grain weight,
biological yield, grain yield and harvest index respectively, with MLE spray at tillering +
jointing + booting + heading. MLE spray only at heading gave 6.84, 3.17, 6.80 and 3.51%
more 1000-grain weight, biological yield, grain yield, and harvest index respectively, as
compared to control. MLE extended the seasonal leaf area duration (Seasonal LAD) by 9.22
and 6.45 d over control when applied at all growth stages and single spray at heading,
respectively. MLE foliar improved salinity tolerance in wheat by improving wheat seedlings
vigour, more chlorophyll a and chlorophyll b contents, exclusion of Na+ and Cl- along with
accumulation of K+ and larger contents of enzymatic antioxidants (catalase, superoxide
dismutase and peroxidase) and non enzymatic antioxidants (ascorbic acid and total phenolic
contents) leading to more 100 grain weight as compared to benzylaminpurine (BAP), hydrogen
peroxide (H2O2) and control (water spray) under salinity. The water sprayed plants under
highest salinity showed maximum accumulation of Na+ and Cl- while largest K+ contents were
observed in case of MLE spray under moderate salinity. MLE and BAP application improved
leaf total soluble protein and superoxide dismutase (SOD). For non-enzymatic antioxidants
(total phenolics and ascorbic acid), MLE was ranked first under moderate salinity. BAP
improved number of grains spike-1 but heavier grains were observed under MLE application in
moderately saline conditions. These effects of MLE were more apparent under moderate
salinity (8dS m-1) as compared to higher salinity (12dS m-1). The water stress caused reduction
in growth and grain yield of wheat due to decreased leaf area and reduced chlorophyll a and
chlorophyll b contents. However, foliar application of MLE and BAP minimized these effects
of drought. MLE application produced more grain yield under moderate and severe water
stress as compared to BAP, K+ and control. Moringa leaves being a rich source of -carotene,
protein, vitamin C, calcium and potassium and act as a good source of natural antioxidants
such as ascorbic acid, flavonoid, phenolics and carotenoid, so the exogenous applications of
MLE improve the antioxidant status and yield of wheat under drought stress. In case of tomato
crop foliar application of MLE30 exhibited large number of flowers, more number of fruits as
well as heaviest fruits. The foliar application of BAP produced same number of flowers but
lighter weight fruits as compared to MLE30. The minimum fruit weight was recorded in case
of foliar applied MLE10, MLE0 and control. The effectiveness of MLE as crop growth
enhancer might be due to the presence of growth promoting substances. MLE proved a
potential growth enhancer in mitigation of abiotic stresses.
xiv

Chapter 1
Introduction
Agriculture is facing the dual challenges of increasing crop production and climate change.
Rising temperature, drought, salinity, floods, desertification and weather extreme are
adversely affecting agriculture especially in developing world (IPCC, 2007). Most of the
predicted population growth to 2030 will be in developing countries (Population Reference
Bureau, 2011) and more than half of the work force engaged in agriculture in the third world
countries is prone to more damage by climate change. Thus, there is need to improve crop
productivity under changed climate, abiotic stresses and to meet the needs of increasing
world populations.
Of various abiotic stresses, high temperature, salt stress and drought alone or in combination
are major threats to crop productivity. Rising temperatures may lead to altered geographical
distribution and growing season of agricultural crops by allowing the threshold temperature
for the start of the season and earlier crop maturity (Porter, 2005). An extreme temperature
shortens the growing period and adversely effects all phases of growth such as tillering,
flowering and grain filling in late sown wheat. The early senescence of leaves results in too
low photosynthetic rate to contribute in fixing carbon to rest of the plant (Hensel et al., 1993;
Sharma-Natu et al., 2006) leading to poor quantity and quality of the harvest (Hussain et al.,
2008). A series of morphological, physiological, biochemical and molecular changes may
reduce expression of full yield potential of crop plants under these climatic stress conditions
(Wang et al., 2001).
The decreased soil water potential causes much reduction in leaf expansion as compared to
root expansion rate under drought or salinity (Kaminek et al., 1997). The water scarcity
disturbs plant metabolic activities by upsetting the membrane structure, altered mineral
uptake (Pospilov et al., 2000), reduction in the chlorophyll contents, relative water
contents and membrane stability index (Tas and Tas, 2007). The limited availability of water
not only reduces number of grains per spike but also decreases the grain weight in wheat (Li
et al., 2000) and quality like low proteins (Garg et al., 2004).

The increased accumulation of Na+ and Cl- under saline conditions leads to the reduced
growth of vascular plants (Munns, 2002). Salt stress causes less germination and poor
seedlings establishment in most of the crops such as wheat (Afzal et al., 2006b), maize,
barley (El-Tayeb, 2005), sugar beet (Ghoulam et al., 2001), sunflower (Ashraf and Tufail,
1995), canola (Athar et al., 2009), cotton and rice (Sattar et al., 2010). Reduction in growth
and yield under salinity is mainly due to salt-induced osmotic stress and specific ion toxities
(Munns and Tester, 2008). The depressed level of natural osmoprotectants and endogenous
hormones are observed in several plants growing in saline soils (Debez et al., 2001).
The combined effect of extreme temperature, drought and salinity includes osmotic damages
(Xiong et al., 2002), oxidative stress like increased generation of reactive oxygen species
(ROS) (Mittler, 2002, Gill and Tuteja, 2010) and protein denaturation (Zhu, 2002).
Biomolecules such as proteins, DNA and lipids are badly injured by reactive oxygen species
(ROS) resulting in denaturation, mutation and peroxidation (Quiles and Lopez, 2004). The
peroxidation of membrane lipids of plasmalemma and other cellular organelles (Candan and
Tarhan, 2003) leads to cell death as a consequence of ROS toxicity.
It has been identified that plants develop many adaptations to cope with stress conditions i.e.
osmotic adjustment, compartmentalization of compatible solutes, alterations in nutrients
ratios specially K+/Na+, evapotranspiration modifications by reducing leaf size, changes in
photosynthetic pigments, stimulation of plant hormones and better antioxidant scavengers
(Sairam and Tyagi, 2004). The breeding programmes to develop crop plants with
aforementioned traits are laborious and time consuming (Javid et al., 2011). The alternative
approaches are management practices including exogenous application of various
antioxidants, mineral elements and plant growth regulators (PGRs). The antioxidants increase
the scavenging capacity against ROS (Mano, 2002). The antioxidants involved in
detoxification of ROS exist in all plants under stress and are categorized as enzymatic such
as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase
(POD), glutathione reductase (GR) and monodehydroascorbate reductase (MDAR) and nonenzymatic i.e. total phenolics (TPC) and ascorbic acid (AA) (Foyer, 2002). The degree to
which the amount and activities of antioxidant enzymes increase under abiotic stress is
extremely variable among several plant species and even between two cultivars of the same
species (Chaitanya et al., 2002). The level of response depends on the species, the
2

development and the metabolic state of the plant, as well as the duration and intensity of the
stress.
Many plants produce significant amount of antioxidants to prevent the oxidative stress
caused by photons and oxygen, they represent a potential source of compounds with
antioxidant activity such as ascorbic acid, total phenols, and vitamins in addition to mineral
elements K+, Ca2+ and PGRs.
Ascorbic acid is an important antioxidant, which reacts not only with H2O2 but also with O2,
OH and lipid hydroperoxidases. In addition to the well established ascorbic acid in animals
against a wide range of ailments and diseases it has been implicated in several types of
biological activities in plants such as an enzyme co-factor, as an antioxidant and as a donor/
acceptor in electron transport at the plasma membrane or in the chloroplasts, all of which are
related to oxidative stress resistance (Conklin, 2001). In chloroplasts ascorbate peroxidase
uses ascorbic acid thereby minimizes the risk of escape and reaction of ROS with each other
near PSI (Foyer and Noctor, 2000). Ascorbate also protects or regenerates oxidized
carotenoids or tocopherol (Imai et al., 1999).
Plant phenolic compounds are also known for their function as antioxidants due to their free
radical-scavenging capabilities (Wattenberg et al. 1980; Barclay et al. 1990; Fauconneau et
al. 1997).
In addition to antioxidants plants also contain a wide range of vitamins that are essential not
only for human metabolism but also for plants, because of their redox chemistry and role as
cofactors, some of them also have strong antioxidant potential. The antioxidant vitamins that
have been the focus of most attention in plants are carotenoids (pro-vitamin A), ascorbate
(vitamin C) and tocochromanols (vitamin E, including both tocopherol and tocotrienols)
(Demmig-Adams and Adams, 2002; DellaPenna and Pogson, 2006; Linster and Clarke,
2008; Foyer and Noctor, 2009: Cazzonelli and Pogson, 2010; Falk and Munne-Bosch 2010;
Me`ne-Saffrane and DellaPenna, 2010).
To combat abiotic stresses and enhance crop yields, application of mineral nutrients is widely
used. Among mineral nutrients calcium is a very important not only for cell wall and
membrane stabilisation, but has also been found to be involved in the regulation of specific
plant responses to environmental stresses (Braam et al., 1996). Cramer et al. (1988) depicted
3

that Ca2+ has alleviating effect on Na+-induced root growth inhibition. Similarly, shortening
of root growth zones was prevented by Ca2+ under saline condition (Bernstein et al., 1993).
Perera et al. (1995) reported that Ca2+ increased the stomatal conductance and restores the
photosynthesis. Transport of water in the root and leaf growing zones was affected with
Na+/Ca2+ ratio (Cramer, 2002). Tyerman et al. (1997) showed that salinity changed the ionic
content and transport within the plants. They concluded that calcium has many regulatory
functions over membrane characteristics and ionic transport in glycophytes and halophytes.
For example, increasing the external Ca2+ concentration decreased the transport of Na+,
thereby reducing Na+ influx in root cells. The cross talk between Ca2+ and ROS has been
studied during defense and growth responses and stomatal closure (Foreman et al., 2003; Hu
et al., 2007; Mori and Schroeder, 2004). Other studies also implicated Ca2+ function both
upstream and downstream of ROS production (Jiang and Zhang, 2003; Hu et al., 2007).
Potassium (K) is the most abundant cation in higher plants. K+ has been the target of some
researchers mainly because it is essential for enzyme activation, protein synthesis and
photosynthesis (Marschner, 1995; Silva, 2004), and it mediates osmoregulation during cell
expansion, stomatal movements, tropisms, phloem solute transport and the maintenance of
cation: anion balance in the cytosol as well as in the vacuole. K+ supply from soil can be rate
limiting for agricultural production under conditions of osmotic and ionic stress.
Proper exogenous application of PGRs along with certain nutrients, antioxidants, organic and
inorganic chemicals has been used to promote plant growth and development for inducing
abiotic and biotic stress tolerance that results in higher economic return (Ashraf and Foolad,
2007; Farooq et al., 2009). Exogenous use of cytokinins improves the crop growth and yield
under normal or stressful environments in a number of crop species (Zahir et al., 2001).
Cytokinin retards the leaf senescence and increased the photosynthetic pigments (Galuszka et
al., 2001). In a field trial with the application of a commercial cytokinin containing product,
cytogen, increase in yields of corn (26.3%), rice (45.8%), pepper (24.4%), cucumber (62.9%)
and cantaloupe (36.8%) has been reported (Mayeux et al., 1983). Priming with cytokinins
like kinetin or benzyl amino purine (BAP) induces physiological adaptation in wheat by
hormonal balance under stress conditions (Iqbal and Ashraf, 2006). BAP delayed the ROSinduced senescence of wheat leaves owing to increased activities of catalase and ascorbate
peroxidase in addition to preventing chlorophyll degradation under oxidative stress (Zavaleta
4

et al., 2007). Under drought less reduction in total soluble protein, chlorophyll and
carotenoids contents were observed by exogenous application of cytokinin like products such
as thidiazuran, BAP, Kartolin 4 and Kartolin 2 (Chernyadev and Monakhova, 2003).
Ashraf and Foolad (2005; 2007) suggested that exogenous application of these compounds as
seed priming or foliar spray enhanced endogenous level and abiotic stress tolerance. These
biologically active substances can modulate plant responses to stress factors. But continuous
use of synthetic chemicals and use of commercially available plant hormones as
osmoprotectants and stimulators of antioxidants to quench ROS is usually not cost effective
and environmentally friendly. Alternatively, toxicological effects of synthetic antioxidants
and consumer preference for natural products have resulted in increased interest in the
application of natural antioxidants (Castenmiller et al., 2002; Kaur and Kapoor, 2001;
Koleva et al., 2002; Pizzale et al., 2002). The search for safe and effective naturally
occurring antioxidants is now focused on edible plants, especially spices and herbs
(Nakatani, 1997). A large number of plants have been screened as a viable source of natural
antioxidants including tocopherols, vitamin C, carotenoids and phenolic compounds which
are responsible for maintenance of health and protection from coronary heart diseases and
cancer (Castenmiller et al., 2002; Kaur and Kapoor, 2001). Such as seaweed extract which is
a natural products possess cytokinin and auxin like properties and can stimulate endogenous
cytokinin activities of plants (Crouch et al., 1990). Another example is humic acid, which has
auxin-like activity, not only enhances plant growth and nutrient uptake but also improves
stress resistance (Zhang and Ervin, 2004). Among different natural sources used to extract
PGRs and antioxidants moringa (Moringa oleifera) is gaining a lot of attention these days
(Foidle et al., 2001).
Moringa is one of the 13 species of genus Moringa and family Moringnance. It is well
known vegetable in Africa, Arabia, India, Southeast Asia, America and Pakistan (Sengupta
and Gupta, 1970). Its roots, fruits, leaves and flowers been used as vegetables (Siddhuraju
and Becker, 2003). Moringa leaves are potential source of vitamin A and C, iron, calcium,
riboflavin, beta-carotene and phenolic acid (Nambiar et al., 2005). Its leaves and oil are a
powerful natural antioxidant (Njoku and Adikwu, 1997). Siddhuraju and Becker (2003)
observed antioxidant properties in the solvent extract of moringa leaves. On the basis of their
results they reported that, moringa leaves are a potential source of natural antioxidants.
5

According to Arabshahi et al. (2007) the extracts from drumstick and carrot had a higher
antioxidant activity (83% and 80%) than -tocopherol (72%). Jongrungruangchok et al.
(2010) compared the composition and mineral contents of moringa leaf obtained from
different regions of Thailand and reported 19.1-28.8, 2.1-2.5, 16.3- 3.9 and 8.5-13.5 percent
of protein, fat, fiber and moisture. The potassium, calcium and iron contents were in range of
1504.2 - 2054.0, 1510.4 - 2951.1 and 20.3 - 37.6 mg / 100 g dry weight basis. Moreover,
moringa leaf extract (MLE) is enriched with zeatin, a purine adenine derivative of plant
hormone group cytokinin (Barciszweski et al., 2000) known for stay green and stress
tolerance capabilities.
It is evident from earlier mentioned reports that MLE possess antioxidants in considerable
amounts but very little published literature is available that explains MLE regulated
metabolic/physiological processes of wheat and other crops subjected to abiotic stress.
In view of all these reports, it is hypothesized that leaf extract from moringa, having a
number of plant growth promoters, mineral nutrients and vitamins in a naturally balanced
composition, may be beneficial for plant growth and development. This study was conducted
to evaluate whether the adverse effects of stress on wheat plants could be mitigated by
exogenous application of osmoprotectants i.e. K+, H2O2, synthetic cytokinin benzyl amino
purine (BAP) and MLE especially focusing the plant antioxidant enzyme system. The
objectives of study were the optimization of MLE dose as natural plant growth enhancer in
comparison to synthetic ones in different crops under normal and abiotic stresses.

Chapter 2
Review of Literature
Plant growth and productivity is regulated by a variety of external and endogenous factors.
Optimum growth and development is ensured when external and internal climatic factors are
within the optimum range. The external factors include different biotic and abiotic stresses.
Among abiotic stresses extreme temperatures (Ferris et al., 1998), drought (Bosch and
Alegre, 2004), salinity (Munns and Tester, 2008), waterlogging (Olgun et al., 2008), low or
high solar radiation (Alexieva et al., 2001), phototoxic compounds (Jamal et al., 2006) and
inadequate availability of mineral nutrients in soil (Fageria et al., 2010) are important. The
endogenous factors comprised of mineral nutrients (Azeem and Ahmad, 2011), plant growth
regulators (Pospilov et al., 2000), antioxidants (Shoresh et al., 2011) and plant water status
(Athar and Ashraf, 2005). It is believed that biotic and abiotic stresses cause a major
reduction in crop yields (Athar and Ashraf, 2009).
Plants responses to these stresses are not simple pathways, but are integrated complex
circuits comprised of multiple pathways and specific cellular compartments, tissues, and the
interaction of additional cofactors and/or signaling molecules to coordinate against an
external stress or stimuli to show a specified response (Dombrowski, 2003).
Therefore a brief review for external stresses like extreme temperature, drought and salinity
along with endogenous factors effecting plant growth and their mitigation is mentioned
below:

2.1. Abiotic stresses


2.1.1. High temperature stress
In many areas of the world heat stress induced by increased temperature is one of major
agricultural problems. The economic yield reduced drastically as a result of morpho
physiological and biochemical changes caused by transient or constant high temperature
which also badly affect plant growth and development.
2.1.1.1. Heat stress
Heat stress is often defined as the rise in temperature beyond a threshold level for a period of
time sufficient to cause irreversible damage in plant growth and development (Wahid et al.,
2007). In general, a transient elevation in temperature, usually 1015C above ambient, is

considered heat shock or heat stress. However, heat stress is a complex function of intensity
(temperature in degrees), duration and rate of increase in temperature. The extent to which it
occurs in specific climatic zones depends on the probability and period of high temperatures
occurring during the day and/or the night. The heat stress as a result of high ambient
temperatures is becoming a serious threat to crop production worldwide (Hall, 2001). A short
exposure to very high temperatures, caused severe cellular injury and even cell death within
minutes (Schoffl et al., 1999) whereas moderately high temperature may take long time to
cause injuries or cell death. The denaturation and aggregation of protein with increased
fluidity of membrane lipids were direct injuries due to high temperatures. Indirect or slower
heat injuries were comprised of enzymes inactivation in mitochondria and chloroplast,
retardation of protein synthesis, protein degradation and loss of membrane integrity
(Howarth, 2005). These injuries eventually lead to starvation, growth inhibition, reduced ion
flux, generation of toxic compounds and reactive oxygen species (ROS) (Schoffl et al.,
1999; Howarth, 2005).
2.1.1.2. Heat stress threshold
The threshold temperature is important in physiological research as well as for crop
production. The value of daily mean temperature at which a detectable reduction in growth
begins referred as threshold temperature. The knowledge of lower and upper developmental
threshold temperatures below and above which growth stops is necessary in physiological
research as well as for crop production. For many plant species upper and lower
developmental threshold temperatures have been determined through experimentation. The
different plant species have different base or lower and upper threshold temperatures but 0C
is often predicted as best base temperature for cool season crops (Miller et al., 2001). The
determination of a consistent upper threshold temperature is difficult due to variation in plant
behavior depending on other environmental conditions (Miller et al., 2001). The threshold
temperature values for tropical crops often higher than temperate or cool season crops.
Threshold temperatures for wheat is 26C at post-anthesis (Stone and Nicolas, 1994), rice
34C at grain yield (Morita et al., 2005), pearl millet 35C at seedling (Ashraf and Hafeez,
2004), corn 38C at grain filling (Thompson, 1986), brassica 29C at flowering (Morrison
and Stewart, 2002), cotton 45C at reproductive (Rehman et al., 2004), tomato 30C at
emergence (Camejo et al., 2005), cool season pulses 25C at flowering (Siddique et al.,

1999), groundnut 34C at pollen production (Vara et al., 2000), and cowpea 41C at
flowering (Patel and Hall, 1990).
2.1.1.2. Heat stress sensitive growth stages
The developmental stage at which the plant is exposed to the stress may determine the
severity of possible damages experienced by the crop. It is, however, unknown whether
damaging effects of heat episodes occurring at different developmental stages are cumulative
(Wollenweber et al., 2003). Vulnerability of species and cultivars to high temperatures may
vary with the stage of plant development, but all vegetative and reproductive stages are
affected by heat stress to some extent. During vegetative stage, for example, high day
temperature can damage leaf gas exchange properties. High temperature induced reduction in
relative growth rate and net assimilation rate along with minimum effects on leaf expansion
were observed in sugar cane (Wahid, 2007), pearl millet and maize (Ashraf and Hafeez,
2004). Moreover, yield of a crop species depends on amount of photosynthetic tissue,
number of leaves, size of leaf or total leaf area. For example, in wheat, yield can be
forecasted upon leaf area index (LAI) at flowering stage as well as crop growth rate (CGR)
(Islam, 1992). The leaf area index (LAI) and crop growth rate (CGR) are determined by
temperature prevails during vegetative growth period (Zhang et al., 1999). While assessing
the effect of late sowing on wheat yield, Farooq et al. (2008) found that prevailing high
temperature reduced LAI and CGR, which resulted in reduced yield.
From these reports mentioned above, it is suggested that increase in ambient temperature
during early or late vegetative growth stage reduced yield indirectly by reducing growth.
Of various plant developmental stages, reproductive stage is most sensitive to temperature
stress. During reproductive stage, flowering, fertilization, and post fertilization processes
markedly affected by high temperatures in most plants, which resulted in reduced crop yield.
For example, a short period of heat stress can cause significant increases in floral buds and
opened flowers abortion (Guilioni et al., 1997; Young et al., 2004). Similarly, impairment of
pollen and anther development by elevated temperatures is another important factor
contributing to decreased fruit set in many crops at moderate to high temperatures (Peet et
al., 1998; Sato et al., 2006). Under high temperature conditions, earlier heading is
advantageous in the retention of more green leaves at anthesis, leading to a smaller reduction
in yield (Tewolde et al., 2006). Heat stress during anthesis induced sterility in many plant

species. It was concluded from controlled conditions experiments that 10-15 days after
flower visibility, 8-9 days prior to anthesis and fertilization were most sensitive for high
temperature stress in various crop plants (Foolad, 2005). The heat stress associated infertility
and yield decline have been observed in many crops such as wheat (Ferris et al., 1998)
cotton, rice (Hall, 1992), pea (Guilioni et al., 1997) and peanut (Vara Parsad et al., 1999). At
the mid-anthesis stage high temperature is also injurious particularly for grain set and grain
fertilization affecting number of grains per spike and grain weight, thereby, leading to low
yield (Ferris et al., 1998; Siddique et al., 1999). This is because under such conditions plants
tend to divert resources to cope with the heat stress and thus limited photosynthates would be
available for reproductive development (Paulsen, 1994; Gifford and Thorne, 1984; Blum et
al., 1994). The reduced fruit set at high temperature stress can be explained in view of the
arguments of Kinet and Peet (1997) who suggested that high temperature caused reduction in
carbohydrates and growth regulators biosynthesis and their translocation in plant sink tissues.
In dwarf wheat variety, high temperature induced decrease in cytokinin content was found to
be responsible for reduced kernel filling and its dry weight (Banowetz et al., 1999). The
improvement in grain yield by BA supply was found with enhanced number of grains
(Trckova et al., 1992) whereas due to increased grain weight rather than grain number
(Warrier et al., 1987; Gupta et al., 2003) under normal as well as late sown conditions. The
heavier grains reflected the more accumulation and partitioning of dry matter towards sinks
(grains) resulting in higher harvest index under BA application during high temperature
stress in late sown wheat (Gupta et al., 2003).
Thus, for successful crop production under high temperatures, it is important to know the
threshold temperature, developmental stages and plant processes that are most sensitive to
heat stress.

2.2.1. Salinity stress


Among abiotic stresses, salinity stress is becoming a major limitation to crop productivity
(Munns, 2011). According to estimates, about 900 Mha land is affected with salt stress
(Flowers, 2004). The presence and accumulation of salts approximately affected 30% of
cultivated soils (Zhu et al., 1997). Excessive amounts of neutral soluble salts such as sodium
chloride (NaCl), sodium carbonate (Na2CO3) and partially calcium chloride (CaCl2) results in
salty soils.

10

2.2.1. Adverse effects of salt stress on plant growth and yield


Salt stress causes adverse effects on plant growth and development by altering physiological
and cellular processes (Greenway and Munns, 1980; Ashraf, 1994; Munns, 2002; 2011;
Munns and Tester, 2008). The reduction of plant growth and dry matter accumulation under
saline conditions has been reported in several important grain legumes (Tejera et al., 2006;
Munns et al., 2006). It is believed that salt stress greatly reduced wheat growth at all
developmental growth stages, particularly at the germination and seedling stage (Munns et
al., 2006). Salt stress causes less germination and poor seedlings establishment in most of the
crops such as wheat, barley, rice, alfalfa (Munns and Tester, 2008).
2.2.1.1. Bases of adverse effects of salinity
The toxic effects of salinity can be visualized from impairment of physiological and
biochemical processes i.e. photosynthesis, protein synthesis and lipid metabolism thereby
reducing plant growth. The salt induced growth reduction is generally attributable to water
deficit or osmotic stress, specific ion toxicities, nutritional imbalances and oxidative stress
(Greenway and Munns, 1980; Munns, 1993; Afzal et al., 2006a; Munns and Tester, 2008).
2.2.1.1.1. Osmotic stress
Reduction in growth and yield under salt stress is mainly due to salt induced osmotic stress
(Munns and Tester, 2008). Excessive amount of soluble salts in the root environment causes
osmotic stress, which may result in the disturbance of the plant water relations in the uptake
and utilization of essential nutrients and also in toxic ion accumulation. As a result of these
changes, the activities of various enzymes and the plant metabolism are affected (Munns,
2002; Lacerda et al., 2003). Reduction in the rate of leaf and root growth is probably due to
factors associated with water stress rather than a salt specific effect (Munns, 2002). The
reduced leaf area observed under highest salinity level may be due to reduction in water
uptake and the nutritional imbalance causing toxicities or deficiencies of ions, so resulting in
leaf injuries (Munns and Tester, 2008). The degree of growth inhibition due to osmotic stress
depends on the time scale of the response, for the particular tissue and species in question,
and whether the stress treatments are imposed abruptly or slowly (Ashraf, 1994; Munns et
al., 2000). Mild osmotic stress leads rapidly to growth inhibition of leaves and stems,
whereas roots may continue to grow and elongate (Hsiao and Xu, 2000).

11

2.2.1.1.2. Specific ion toxicities


Besides salt induced osmotic stress, accumulation of toxic salts also causes growth
suppression in plants. Dominant salts found in salt affected soils are generally Na+, Cl-, SO4 2and HCO3 which result in severe toxicity. However, the crops show variations in their
sensitivities to different toxic ions. It is generally believed that excessive accumulation of
Na+ causes nutrient imbalance, thereby resulting in specific ion toxicity (Greenway and
Munns, 1980; Grattan and Grieve, 1999). In salt sensitive species or at higher salinity levels,
plant loose its ability to control Na+ transport as a result salt induced ionic effect dominates
the osmotic effect (Munns and Tester, 2008). In most species, accumulation of Na+ to a toxic
level appears to occur more rapidly than Cl-, therefore most studies focused on Na+ exclusion
and that of Na+ transport control within the plant (Munns and Tester, 2008). For example,
more accumulation of Na+ and Cl- was observed in all parts of guava, especially in the leaves
thereby resulting in reduced growth (Ferreira et al., 2001). In addition to Na+ being a toxic
ion, Cl- is considered to be the more toxic ion in some species such as soybean, citrus, and
grapevine (Lauchli, 1984; Grattan and Grieve, 1999; Storey and Walker, 1999). Thus, the
accumulation of any cation or anion in excessive amounts in growth medium can cause
toxicity and growth reduction depending upon species or cultivar.
2.2.1.1.3. Nutritional imbalances
The interactions of salts with mineral nutrients may result in considerable nutrient
imbalances and deficiencies (McCue and Hanson, 1990). Ionic imbalance occurs in the cells
due to excessive accumulation of Na+ and reduces uptake of other mineral nutrients such as
K+, Ca2+, and Mn2+ (Karimi et al., 2005). High sodium to potassium ratio due to
accumulation of high amounts of sodium ions inactivates enzymes and affects metabolic
processes in plants (Booth and Beardall, 1991). Excess Na+ and Cl- inhibits the uptake of K+
and leads to the appearance of symptoms like those in K+ deficiency. The deficiency of K+
initially leads to chlorosis and then necrosis (Gopal and Dube, 2003). The role of K+ is
necessary for osmoregulation and protein synthesis, maintaining cell turgor and stimulating
photosynthesis (Freitas et al., 2001). Both K+ and Ca2+ are required to maintain the integrity
and functioning of cell membranes (Wenxue et al., 2003). Maintenance of adequate K+ in
plant tissue under salt stress seems to be dependent upon selective K+ uptake and selective
cellular K+ and Na+ compartmentation and distribution in the shoots (Munns et al., 2000;

12

Carden et al., 2003). The maintenance of calcium acquisition and transport under salt stress
is an important determinant of salinity tolerance (Soussi et al., 2001; Unno et al., 2002). Salt
stress decreases the Ca2+/Na+ ratio in the root zone, which affects membrane properties due
to displacement of membrane associated Ca2+ by Na+, leading to dissolution of membrane
integrity and selectivity (Kinraide, 1998). The increased levels of Na+ inside the cells change
enzyme activity resulting in cell metabolic alteration. Disturbance in K+ uptake and
partitioning in the cells and throughout the plant may even affect stomatal opening thus
diminishing the ability of the plant to grow. Externally supplied Ca2+ has been shown to
ameliorate the adverse effects of salinity on plants, presumably by facilitating higher K+/Na+
selectivity (Hasegawa et al., 2000). Another key role attributed to supplemental Ca2+ addition
is its help in osmotic adjustment and growth via the enhancement of compatible organic
solutes accumulation (Girija et al., 2002). From above discussion it is clearly evident that
reduced accumulation of Ca2+, K+ and Mg2+ with increased uptake and accumulation of Na+
greatly reduced crop growth and yield under salt stress.
2.2.1.1.4. Oxidative stress
Salt stress causes oxidative stress in plants i.e., the production of reactive oxygen species
(ROS) such as H2O2 (hydrogen peroxide), 1O2 (singlet oxygen) and OH- (hydroxyl radical).
Many biomolecules such as proteins, DNA and lipids are badly injured by ROS resulting in
cell death (Apel and Hirt, 2004). The decrease in the content and the activity of various
antioxidants in response to salt stress has been reported in several species (Shalata and
Neumann, 2001; Al-Hakimi and Hamada, 2001; Athar et al., 2008) and is regarded as one of
the mechanism explaining, at least in part, the deleterious effects of salinity on crops. Plants
use two systems to defend against and repair damage caused by oxidizing agents. First, the
enzymatic antioxidant system which is mainly represented by superoxide dismutase (SOD),
peroxidase (POD), catalase (CAT) and ascorbate peroxidase (ASPX) (Harinasut et al., 1996),
then, the non-enzymatic

antioxidant system, consisting of molecules involved in ROS

scavenging such as ascorbic acid (vitamin C), alpha-tocopherol (vitamin B), -carotene,
glutathione (tripeptide) (Piotr and Klobus, 2005). Several studies have demonstrated the
existence of a close positive correlation between the rate and extent of the increase in
antioxidant activity and plant salt tolerance (Sairam et al., 2005). The significant decrease in
cytokinin concentration was observed in roots and shoots of barley cultivars under salinity

13

(Goicoechea et al., 1995). According to Zhang and Ervin (2008) the improvement in stress
tolerance of creeping bent grass with increased SOD level has been observed by the
application of cytokinin containing sea weed extract (SWE).
It becomes clearly evident that antioxidant status of plant for scavenging of ROS is an
important salt tolerant trait.
2.2.1.2. Crop growth stage and salt stress
The plants differ in their extent of salt tolerance at different stages of vegetative and
reproductive growth. Germination is the most crucial stage in this regard (Ahmad and
Jabeen, 2005). Seed often fails to germinate in salt affected soils. The reduction in rate of
seed germination and final germination percentage were observed under high salt contents
(Fowler, 1991). Almost all stages of growth and development, flowering and fruit set were
adversely affected by salinity, thereby, causing low economic yield and poor quality of
production (Ashraf and Harris, 2004).
2.2.1.3. Salinity and photosynthetic capacity
The yield of a crop is mostly affected by its photosynthetic ability (Akram et al., 2002) which
is one of the main contributing factors in salt induced reduction in plant growth and yield.
Tolerance of the photosynthetic system to salinity depends on how effectively a plant
excludes or compartmentalizes the toxic ions. Plant characteristics such as leaf surface area,
CO2 assimilation and yield negatively correlated with salinity. According to Hanaa et al.
(2008) considerable decease in chlorophyll a and b contents were observed in wheat plants
irrigated with sea water as compared to normal water irrigated plants. It may be attributed
that more Na+ at higher salinity enhanced degradation of chlorophyll or decreased its
biosyntheses by lowering Mg2+, an important constituent of chlorophyll (Rubio et al., 1995).
Salt stress enhances the accumulation of NaCl in chloroplasts and is often associated with
decrease in photosynthetic electron transport activities (Kirst, 1989). In higher plants, salt
stress inhibits photosystem (PS-II) activity (Kao et al., 2003), although some studies showed
contrary results (Brugnoli and Bjrkman, 1992).
From these reports it is summarized that salt induced growth inhibition may be resulted from
reduction in photosynthetic capacity. A number of factors such as photosynthetic pigments,
amount of photosynthesizing tissue or leaf area, gas exchange and metabolism etc. affect the

14

photosynthetic capacity. Plants tolerance to salinity depends on how the photosynthetic


machinery may be protected from osmotic and toxic effects of salt stress.

2.3.1. Drought stress


Global climate change makes drought a serious threat to food security world wide (Elisabeth
et al., 2009). Crop productivity is determined by the total amount of precipitation and also by
its distribution during the growing season (Loss and Siddique, 1994; Slafer, 2003). It has
been estimated that the countries that generate two-third of the worlds agricultural product
experience water deficit conditions on a regular basis (Revenga et al., 2000). For instance,
China, Australia, many African and South American countries, the Middle East, Central
Asia, and many states of the United States often experience severe drought conditions
(http://www.globalresearch.ca/index.phpcontextva&aid12252), resulting in significant
decline in food grain production. The projected changes in climate in the coming years may
exacerbate the adverse effects of drought not only in food crops but also in other
economically important crops. The severity of drought is unpredictable as it depends on
many factors such as occurrence and distribution of rainfall, evaporative demands and
moisture storing capacity of soils (Wery et al., 1994).
A brief review of drought induced changes in crop plants is mentioned below:
2.3.1.1. Osmotic stress
Exposure of plants to drought stress substantially decreases the leaf water potential, relative
water content and transpiration rate, with a concomitant increase in leaf temperature
(Siddique et al., 2001). The ratio between dry matter produced and water consumed is termed
as water use efficiency at the whole-plant level (Monclus et al., 2005). Abbate et al. (2004)
concluded that under limited supply, water use efficiency of wheat was greater than in wellwatered conditions. They correlated this higher water-use efficiency with stomatal closure to
reduce the transpiration. Drought causes a significant reduction in plant water content and
cell turgor potential, which in most cases results in reduced growth rate and final crop yield.
2. 3.1.2. Nutrient stress
An important effect of water deficit is on the acquisition of nutrients by the root and their
transport to shoots. Lowered absorption of the inorganic nutrients can result from
interference in nutrient uptake and the unloading mechanism, and reduced transpirational
flow (Garg, 2003; McWilliams, 2003). The nutrients uptake by the plants were reduced under

15

drought stress conditions, the nitrogen and phosphorus being stored in grains while K
accumulation sites were stem and leaves. 67 and 82 % reduction was observed in K uptake
under mild and severe water stress conditions (Baque et al., 2006). The insufficient K supply
induced reduction of photosynthesis under drought stress (Cakmak, 2005). As K played a
role in CO2 fixation during photosynthesis so the plants suffering from drought exhibit more
K requirements (Cakmak and Engels, 1999). If K become deficient under drought stress
additional disturbances were observed in water relations, stomatal movements and
photosynthesis (Marschner, 1995).
2.3.1.3. Oxidative stress
Similar to many other stresses, drought can cause oxidative stress in plants, under which
reactive oxygen species (ROS) such as superoxide radical (O-2), hydroxyl radical (OH-),
hydrogen peroxide (H2O2), and alkoxy radical (RO) are produced (Munne-Bosch and
Penuelas, 2003). ROS can damage cell membranes, nucleic acids, and proteins (Ashraf,
2009; Mittler, 2002), causing metabolic imbalances in plants. A linear relationship exists
between generation of reactive oxygen species and severity of drought, which enhanced
membrane lipids peroxidation, disintegration of nucleic acid and degradation of proteins
(Farooq et al., 2009). Simultaneously, plants produce a variety of antioxidants that counteract
the generation of ROS in response to drought stress (Munne- Bosch and Penuelas, 2003;
Wang et al., 2009). A reduction in the activities of superoxide dismutase, peroxidase,
catalase and protein contents in leaves of maize crop towards the physiological maturity were
observed under water stress (Ti-da et al., 2006). The enhanced activities of SOD, CAT and
POD in maize leaves were observed during light water stress and then reduced under severe
stress but their levels were even better than fully irrigated plants (Ti-da et al., 2006).
2.3.1.4. Hormonal imbalance
Drought stress often leads to hormonal imbalances (Bajguz and Hayat, 2009; Farooq et al.,
2009), changes in activities of enzymes responsible for regulation of key metabolic processes
(Turkan et al., 2005) and modulation of signal transduction (Chaves et al., 2003), gene
expression (Denby and Gehring, 2005), respiration (Ribas-Carbo et al., 2005), and
photosynthesis (Flexas et al., 2004). The hormonal status of plant cells might be balanced by
addition of cytokinin during water stress (Banowetz, 1998). The chlorophyll contents were
significantly improved by exogenous application of growth regulators under water scarcity

16

conditions (Zhang et al., 2004). In the presence of cytokinin like activity compounds e.g.
Kartolin less reduction was observed in protein contents (Chernyadev and Monakhova,
2003). The positive effects of BAP and TDZ on protein pool were also pronounced under
drought stress. The phytoregulators exhibiting cytokinin activity show protective effects in
water deficit and prevent reduction in chlorophyll and protein contents. The dehydration
stress drastically decreases the cytokinin level. The decreased contents under drought stress
may be due to reduced cytokinin biosyntheses or its enhanced degradation (Pospilov et al.,
2000). The lesser quantities of cytokinin was also observed in xylem sap under drought stress
(Dodd, 2003) emphasized the need for exogenous application of cytokinin.
2.3.1.5. Crop growth stage and drought stress
The reduced germination and seedling stand is the first and foremost effect of drought (Kaya
et al., 2006). In a study on pea, drought stress impaired the germination and early seedling
growth of five cultivars tested (Okcu et al., 2005). Moreover, in alfalfa (Medicago sativa),
germination potential, hypocotyl length and shoot and root fresh and dry weights were
reduced by polyethylene glycol induced water deficit, while the root length was increased
(Zeid and Shedeed, 2006). Water deficit during pollination increased the frequency of kernel
abortion in maize (Zea mays). In pigeon pea, drought stress coinciding with the flowering
stage reduced seed yield by 4055% (Nam et al., 2001) Post-anthesis drought stress was
detrimental to grain yield regardless of the stress severity (Samarah, 2005). Following
heading, drought had little effect on the rate of kernel filling in wheat but crop duration (time
from fertilization to maturity) was shortened and caused dry weight reduction at maturity
(Wardlaw and Willenbrink, 2000). This yield reduction was due to reduced grain filling
duration rather than grain filling rate (Wardlaw and Willenbrink, 2000).Water stress applied
at pre-anthesis reduced time to anthesis while at post anthesis it shortened the grain filling
period in triticale genotypes (Estrada-Campuzano et al., 2008) and badly effected grain yield
irrespective of its severity (Samarah, 2005).
2.3.1.6. Photosynthesis and drought stress
Drought stress produced changes in photosynthetic pigments and components (Anjum et al.,
2003), damaged photosynthetic apparatus (Fu and Huang, 2001) and diminished activities of
calvin cycle enzymes which are important causes of reduced crop yield (Monakhova and
Chernyadv, 2002). The leaf senescence visualized initially by leaf yellowing due to

17

chlorophyll degradation and decrease in the ratio of chlorophyll to carotenoids has been
observed during drought induced leaf senescence in many field crops (Yang et al., 2002). A
13-15 % reduction in chlorophyll content was observed in wheat seedlings on 7th day
followed by drought exposure (Nikolaeva et al., 2010). The chlorophyll a, b and leaf soluble
protein contents were decreased under osmotic stress conditions (Singh et al., 1998). Water
defiency caused a 30-40% reduction in soluble protein contents of adult leaves (Chernyadev
and Monakhova, 2003). Another important effect that inhibits the growth and photosynthetic
abilities of plants is the loss of balance between the production of reactive oxygen species
and the antioxidant defense (Fu and Huang, 2001; Reddy et al., 2004).
2.3.1.7. Crop yield and drought stress
Drought stress adversely affects a variety of vital physiological and biochemical processes in
plants, leading to reduced growth and final crop yield. Reduction in yield depends on water
stress severity, duration and plant developmental stage at which plant experience water stress
(Plaut, 2003). In barley, drought stress reduced grain yield by reducing the number of tillers,
spikes, grains per plant and individual grain weight. In maize, water stress reduced yield by
delaying silking thus increasing the anthesis to silking interval. This trait was highly
correlated with grain yield, specifically ear and kernel number per plant (Cattivelli et al.,
2008). Drought stress in soybean reduced total seed yield and the branch seed yield
(Frederick et al., 2001). In pearl millet (Pennisetum glaucum), co-mapping of the harvest
index and panicle harvest index with grain yield revealed that greater drought tolerance was
achieved by greater partitioning of dry matter from stover to grains (Yadav et al., 2004).
Moisture deficit reduced cotton (Gossypium hirsutum) lint yield, due to reduced boll
production as a result of fewer flowers and greater boll abortions (Pettigrew, 2004). In rice,
on the other hand, water stress imposed during the grain filling period enhanced
remobilization of pre-stored carbon reserves to grains and accelerated grain filling (Yang et
al., 2001). The prevailing drought reduces plant growth and development, leading to
hampered flower production and grain filling and thus smaller and fewer grains. A reduction
in grain filling occurs due to a reduction in the assimilate partitioning and activities of
sucrose and starch synthesis enzymes.

18

2.2. Mitigation of abiotic stresses


Abiotic stresses may cause yield reduction as much as 50% in most major crop plants (Bray
et al., 2000). Extreme temperatures, drought, salinity and oxidative stress are often
interrelated and may cause cellular damage (Wang et al., 2003). Abiotic stresses altered
levels of plant growth regulators (PGRs) resulted in decreased plant growth (Morgan, 1990).
Plants acclimatized to these abiotic stresses by certain physiological and biochemical
modifications. Abiotic stress tolerance in crop plants is associated with osmotic adjustment,
ion exclusion, maintenance of water status, higher nutrient acquisition ability, higher plant
hormone concentrations, higher photosynthetic capacity or antioxidant capacity (Athar and
Ashraf, 2009). Based on this information, various scientists suggest a number of strategies to
manage or improve abiotic stress tolerance in plants. Of various cultural practices or
management practices, early sowing to avoid high temperature, shortage of water at
reproductive stage is most important. Water stress at later phases of grain filling were due to
shortage of assimilate but if vegetative phase of plants extended and thus increased the
duration of photosynthesis, it provides more photoassimilates to grain that resulted in
improved the grain weight. Duration of vegetative phase or active photosynthetic period can
be managed by adjusting time of planting.
Similarly, if considerable amount of genetic variability exists in a crop species, some abiotic
stress tolerant individuals can be selected. Moreover, through conventional breeding, abiotic
stress tolerant traits can be introgressed in stress sensitive cultivars. In addition, with the
advancement of molecular biology techniques, genes for specific traits can also be
transformed in crops to improve stress tolerance (Wang et al., 2003; Ashraf et al., 2008;
Athar and Ashraf, 2009). The serious complexities in screening and conventional breeding
result in slow pace in development of stress resistance methods and pose a major limitation
in use of theses methodologies for induction of stress tolerance.
Since stress tolerant plants had higher compatible solutes, nutrients, plant hormones,
antioxidant compounds than those of stress sensitive cultivars, Ashraf and Foolad (2005;
2007) suggested that exogenous application of these compounds as seed priming or foliar
spray enhanced endogenous level and abiotic stress tolerance. These biologically active
substances can modulate plant responses to stress factors. These strategies are discussed
below:

19

2.2.1. Seed priming


Seed priming is an effective tool to minimize time between germination and emergence
which resulted in synchronized emergence (Harris et al., 2002). In some earlier studies it has
been observed that seed priming with plant growth regulators, inorganic salts, compatible
solutes or sugar beet extract caused improvement in seed germination by providing
physiological and biochemical adaptations (Pill and Savage, 2008; Afzal et al., 2006a). It is
largely known that higher or enhanced mobilization of metabolites/inorganic solutes to
germinating plumule/embryo result in enhanced growth (Taiz and Zeiger, 2002). An
improvement in seedling vigor was observed with CaCl2 and CaCl2 + NaCl priming in
greenhouse conditions (Ruan et al., 2002). Moreover the higher accumulation of Na+ and Clwere also observed in seedlings raised from CaCl2 or NaCl primed seeds. The salinity
decreased the protein contents but remarkable increase in protein content was obtained from
CaCl2 seed priming (Afzal et al., 2006b) which may be due to better defense of membrane
and membrane bound enzymes. Moreover the highest value of 1000 kernel weight in rice
was produced by KCl followed by CaCl2 seed priming (Farooq et al., 2006). Similarly,
priming with cytokinins like kinetin or benzyl amino purine (BAP) increased salt tolerance in
wheat at seedling stage (Iqbal and Ashraf, 2006). In another study with pigeon pea, the
adverse effects of salinity on germination were mitigated by seed priming with kinetin and
ascorbic acid (Jyotsna and Srivastava, 1998). Plant stress tolerance can be increased by seed
priming with cytokinin (Iqbal et al., 2006). The negative effects of NaCl on wheat
chlorophyll contents can be alleviated by application of benzyl amino purine (Mumtaz et al.,
1997).
2.2.2 Foliar application
2.2.2.1 Exogenous foliar application of nutrients
In an extensive review on the role of mineral nutrients in improving stress tolerance, Grattan
and Grieve (1999) stated that foliar applications of those nutrients which become deficient
under stress conditions improved the nutrient status of plants thereby leading to increased
stress tolerance. For example, potassium has been extensively used as a foliar spray to
enhance crop salt tolerance (Ashraf et al., 2008). For increased active uptake of K+ by the
guard cells exogenous application of K+ was required (Premachandra et al., 1993) under
stress conditions. Ahmad and Jabeen (2005) found that foliar spray of plants with potassium

20

containing material minimized the antagonistic effects of soil salinity. The K+ uptake was
improved by increasing application of K+ under water scarcity (Baque et al., 2006). In
tomato, sunflower and lemongrass an increased vegetative growth, total nitrogen and total
carbohydrate content were observed by foliar application of vitamin (thiamine) (AbdelHalim, 1995 Tarraf et al., 1999 and Gamal El-Din, 2005). Akram et al. (2007) found that
foliar spray of various inorganic salts of K+ in sunflower caused considerable improvement
in the ion homeostatic conditions and plant photosynthetic activity through stomatal
movement. Moreover, the extent of stress ameliorative effect of K+ salts thereby growth
enhancement depends upon plant developmental stage of foliar application, salt
concentration and accompanying anion in a specific salt. Thus, foliar application of mineral
nutrients can be beneficial to improve crop salt tolerance.
2.2.1.2 Exogenous foliar application of PGRs
The other effective approach is the exogenous application of plant growth regulators (PGRs)
involved in promoting plant growth and development under normal as well as stressful
conditions (Brathe et al., 2002). Many attempts have been made previously for exogenous
application of cytokinin (natural or synthetic) to enhance grain cytokinin content and the
increment in number of grains and final grain weight was based upon growth stage i.e. time
of application (Wang et al., 2001; Gupta et al., 2003; Yang et al., 2003). According to Gupta
et al. (2003) during post anthesis temperature stress the exogenous application of benzyl
adenine increased grain weight only in heat tolerant wheat genotypes under late sown but in
all genotypes irrespective of their tolerance under normal sowing conditions. The significant
increment in grain weight of wheat by attracting more assimilates towards the developing
grain was observed with the application of benzyl adenine at anthesis (Warrier et al., 1987).
Moreover more grain weight was observed when BAP was sprayed on ears i.e. at
physiological maturity (Hosseini et al., 2008). In soybean not any modification in plant
growth trait was observed by foliar application of cytokinin during vegetative growth period
(Leite et al., 2003). The chlorophyll contents, total protein and tomato yield were increased
under foliar spray of cytokinin (Nasr, 1993). Shahbaz et al. (2008) observed that foliar
applied brassinosteroids counteracted the adverse effects of salt stress on growth of wheat but
did not improve the grain yield. Similarly, foliar applied salicylic acid counteracted the NaCl
deleterious effects on sunflower (Noreen and Ashraf, 2008).

21

2.2.1.3. Exogenous application of antioxidants


Ascorbic acid a potent antioxidant, the foliar application of ascorbic acid minimized the
reduction of chlorophyll a by NaCl in wheat (Khan et al., 2006). Foliar application of
ascorbic acid enhanced accumulation of macronutrient (N, P and K) in sweet pepper (Talaat,
2003). Emam and Helal (2008) found that foliar spray of ascorbic acid recovered the nongerminating flax seeds under saline conditions. The reduction in activities of antioxidants
enzymes superoxide dismutase (14.82%), catalase (31.84%) and peroxidase (26.34%) were
observed under abiotic stresses but the foliar spray of CaCl2 and salicylic acid not only
improved protein content but also showed enhancements in antioxidant enzyme activities
indicating positive regulatory effect of CaCl2 and salicylic acid on antioxidant defense
system in tomato leaves (Li et al., 2009).
2.2.3. Sources of nutrients, PGRs and antioxidants
The exogenous use of commercially available mineral nutrients, antioxidants and cytokinin
to alleviate the adverse effects of abiotic stresses on growth is expensive. So there is a need
to explore the natural sources of PGRs for exogenous use such as algae extract (Hanaa et al.,
2008), humic acid (HA), seaweed extract (SE) (Zhang and Ervin, 2008) and extract obtained
from moringa (Moringa oleifera) leaves (Foidle et al., 2001).
According to Duval and Shetty (2001) the total phenolic contents in green pea shoots were
enhanced under foliar spray of high cytokinin containing anise root extracts combined with
yeast extract.
2.2.3.1. Microalgae
Microalgae

constitute

potential

source

of

antioxidant,

vitamins,

carotenoids,

polysaccharides, phycobiliprotein and posses immune-modulating agent properties (El-Baz,


et al., 2002; Abd El-Baky, et al., 2003 and 2004). It can be used widely in medicine,
industry, marine culture and in combating pollution (Thajuddin and Subramanian, 2005). The
presence of auxin, cytokinins, gibberellins and other growth regulating substances proved it a
plant growth stimulating agent (rdg et al., 2004). For instance, seaweed extracts (SE) have
been used as a cytokinin-like growth regulator and exhibit multiple functions including
stimulation of shoot growth and branching (Temple and Bomke, 1989), increase root growth
and lateral root development (Metting et al., 1990), improve nutrient uptake (Yan, 1993),
enhance resistance to diseases (Featoyby-Smith and Staden, 1983) and environmental

22

stresses such as drought and salinity (Nabati et al., 1994). Proper application of SE-based
cytokinins may be an effective approach to improve summer performance of creeping bent
grass. Application of SE enhanced SOD activity, photosynthetic capacity and chlorophyll
content in tall fescue, especially at 4 weeks after treatment. The more increment in
chlorophyll a and b were observed under foliar spray of algal extract as compared to foliar
spray of bioregulator i.e. ascorbic acid and benzyl adenine in saline conditions (Hanaa et al.,
2008). Moreover, the antioxidant status of wheat plant was positively correlated with
antioxidant level of algal extract.
2.2.3.2. Humic acid
The foliar spray of humic acid not only improved growth and nutrients uptake of some crops
but also enhanced their yields (Padem et al., 1999; Neri et al., 2002). Humic acid nutrient
composition leads to a 25% reduction in soil applied NPK fertilizer requirement with
increase in uptake of N, P, K, Ca, Mg, Fe, Mn, Zn and Cu nutrients by the crop resulting to
enhanced yield (Shaaban et al., 2009). According to Neri et al., (2002) the wetting action and
slow speed of droplet drying make the humic acid best suited for foliar spray. The
significantly higher nutrient contents were observed in leaves as compared to control under
foliar spray of humic acid (Guvenc et al., 1999). The significant positive effects of humic
acid application were found on yield of faba bean and chlorophyll contents of soybean
(Shuixiu and Ruizhen, 2001). 21-38% more root mass was observed in creeping bent grass
by foliar application of cytokinin containing seaweed extract + humic acid under drought
stress (Zhang and Ervin, 2004).
2.2.2.3. Moringa oleifera
The "Moringa" tree is grown mainly in semi-arid, tropical, and subtropical areas. It is
native to the sub-Himalayan tracts of India, Pakistan, Bangladesh and Afghanistan (Fahey,
2005). It grows best in dry sandy soil; it tolerates poor soil including coastal areas. It is a fast
growing, drought resistant tree. Today it is widely cultivated in Africa, Central and South
America, Sri Lanka, India, Mexico, Malaysia, Indonesia and the Philippines. Moringa is a
short, slender, deciduous, perennial tree about 10 m tall with drooping branches, brittle stems
and branches, corky bark, feathery pale green 3060 cm long compound leaves, with many
small leaflets which are 1.32 cm long, 0.60.3 cm wide, fragrant white or creamy-white
flowers having 2.5 cm in diameter and borne in sprays, pendulous brown triangular pods,

23

splitting lengthwise into 3 parts when dry, containing about 20 dark brown seeds embedded
in the pith, pod tapering at both ends. Main root is thick (Foidle et al., 2001). It produces fruit
between April to June in Pakistan. It is considered one of the worlds most useful tree, as
almost every part of the Moringa tree can be used for food or has some other beneficial
property. In the tropics, it is used as forage for livestock, and in many countries moringa
micronutrient liquid, a natural anthelmintic (kills parasites) and adjuvant (to aid or enhance
another drug) is used as a metabolic conditioner to aid against endemic diseases in
developing countries (Foidle et al., 2001). Moringa oleifera is the most nutrient rich plant yet
discovered. Moringa provides a rich and rare combination of nutrients, amino acids,
antioxidants, antiaging and anti-inflammatory properties used for nutrition and healing.
M. oleifera is a miracle tree with a great indigenous source of highly digestible proteins, Ca,
Fe and vitamin C (Fahey, 2005). Some articles and research studies have reported that the
dry leaves of M. oleifera contain 7 times more vitamin C than orange, 10 times vitamin A
than carrot, 17 times calcium than milk, 15 times potassium than bananas, 25 times iron than
spinach and 9 times proteins than yogurt (Fuglie, 1999). In addition, it contains vitamin Bcomplex, chromium, copper, magnesium, manganese, phosphorus and zinc (Fuglie, 2000).
Thurber and Fahey (2009) stated M. oleifera leaves as rich protein source, which can be used
by doctors, nutritionists and community health conscious persons to solve worldwide
malnutrition or under nutrition problems. According to researchers moringa has the potential
to combat vitamin A and other micronutrient deficiencies (Nambiar, 2006). 40139 g/100g
total carotenoides on fresh weight basis in moringa leaves of which 47.8% or 19210 g/100g
was -carotene. Ascorbic acid at 6.6 mg/g on dry weight basis, 0.26 mg/g Fe, 22.4 mg/g
calcium, 6.3 mg/g P, 11.2 mg/g oxallic acid and 0.9 g/100 g fiber. Moringa has been in use
since centuries for nutritional as well medicinal purposes. Another important point is that
Moringa leaves contain all of the essential amino acids, which are the building blocks of
proteins. It is very rare for a vegetable to contain all of these amino acids. Moringa contains
these amino acids in a good proportion, so that they are very useful to our bodies. Given its
nutritional value, it can be utilized in fortifying sauces, juices, spices, milk, bread, and most
importantly, instant noodles. Many commercial products like Zija soft drink, tea, and
neutroceuticals are available all over the globe.

24

2.2.2.3.1 Moringa leaf extract (MLE)


A plant growth spray made from moringa leaves increased crop production 20-35%. Spray
affects the crops by longer life-span, heavier roots, stems and leaves, produce more fruit,
larger fruit and increase in yield 20-35% (Foidle et al., 2001), highlighting its opportunity of
use as a foliar spray to accelerate growth of young plants. MLE proved an ideal plant growth
enhancer in many experiments (Makkar and Becker 1996; Nouman et al., 2011). Makkar et
al. (2007) found the moringa leaves as a source of plant growth factors, antioxidants, carotene, vitamin C, and antioxidant agents. Siddhuraju and Becker (2003) studied the
antioxidants properties of moringa leaf extract and demonstrated that it: (1) reduced
potassium ferricyanide, (2) scavenged superoxide radicals, (3) prevented the peroxidation of
lipid membrane in liposomes, (4) could donate hydrogen and scavenge radicals. Cai et al.
(2004) observed positive correlation between total phenolic contents and antioxidant
activities of methanolic as well as aqueous extracts of Chinese medicinal plants. The aqueous
extract obtained from Andrographis paniculata leaves showed more phenolic content and
antioxidant potential as compared to extract from other parts such as stem and fruits (Arash
et al., 2010). Makkar et al. (2007) found that MLE contains significant quantities of calcium,
potassium, and cytokinin in the form of zeatin, antioxidants proteins, ascorbates and phenols.
MLE priming in rangeland grass Echinochloa crusgalli showed encouraging results and
significantly increased the shoot vigour along with improved number of leaves and fertile
tillers (Nouman et al., 2011). MLE was used as priming agent in hybrid maize. Seed primed
with moringa leaf extract (MLE) diluted to 30 times with tap water increased the germination
speed and spread and seedling vigor under cool conditions (Noman, 2008). According to
Mehboob (2011) high temperature at planting delayed the seedling emergence in control
while seed priming treatments resulted in earlier and vigorous seedling stand. Among all the
strategies, osmopriming with MLE diluted 30 times reduced mean emergence time (MET)
(8.967 vs. 9.097 days) and increased final emergence (FEP) (83.33 vs. 86.333) under
optimum as well late planted conditions as compared to control. Agronomic and yield related
traits were significantly affected by seed priming at both sowing dates. Maximum number of
grains rows per cob (34.933 vs. 31.500), total kernel rows per cob (14.30 vs 13.63) and
higher number of grains per cob (1271.0 vs 1114.0) were recorded for MLE priming.

25

Similarly improved biological (66.75 vs 60.53 t ha-1) and economical yield (6.97 vs 6.23 t ha1

) were recorded for osmopriming with MLE under both optimum and delayed planted

conditions. Increased yield by MLE priming was attributed to enhanced seedling emergence,
chlorophyll contents and cell membrane permeability. The foliar application of MLE and
BAP may also stimulate earlier cytokinin formation thus preventing premature leaf
senescence and resulting in more leaf area with higher photosynthetic pigments (Hanaa et al.,
2008; Rehman and Basra, 2010).
Abiotic stresses severely reduced the crop productivity world wide thus these becoming a
major threat for food security. Based on physiological and molecular mechanisms of abiotic
stress tolerance, various strategies have been advised to improve crop tolerance against
abiotic stresses including screening, selection, breeding and genetic engineering etc.
However, based on physiological and biochemical bases of stress tolerance in crops,
scientists suggested exogenous use of compatible solutes, antioxidants compounds, mineral
nutrients, and plant growth regulators as a shotgun approach. Since synthetic compatible
osmolytes, antioxidants and plant growth regulators are costly, use of plant extracts having
appreciable amount of these compounds could be an economically viable strategy. Among
naturally occurring plant growth enhancers, Moringa oleifera has attained enormous
attention because of having cytokinin, antioxidants, macro and micro nutrients in its leaves.
Therefore, in the present study, moringa leaf extract was exogenously applied to assess up to
what extent it can improve abiotic stress tolerance in some potential crops such as wheat,
tomato and pea. MLE applied through seed priming and foliar application to identify the
effective mode of application. Its secondary objective was to evaluate the dose of exogenous
application of MLE under normal as well as stressful conditions especially focusing the plant
antioxidant enzyme system.

26

CHAPTER 3
MATERIAL AND METHODS
The study was conducted to evaluate the efficacy of Moringa oleifera leaf extract (MLE) as a
natural crop growth enhancer. The modes of application were seed treatment, soil and foliar
application on wheat, peas and tomato crops under normal and abiotic stress conditions. The
experiments were conducted under laboratory, wire house and field conditions. The details
regarding experimental site and materials used during course of these studies are described
here in general whereas the specific methodology is discussed under particular experiments.

3.1. Source of moringa leaves


Young leaves / branches of moringa were harvested from young full grown trees located at
the experimental nursery area of Department of Forestry, Range Management and Wildlife,
University of Agriculture, Faisalabad.

3.2. Analysis of moringa leaves


The oven dried moringa leaves were analysed for their nutrient contents i.e. total nitrogen
(Ryan et al., 2001; Jackson, 1962), phosphorous, potassium, calcium, magnesium, copper,
iron, manganese and zinc (Ryan et al., 2001) and boron (Gaines and Mitchell, 1979;
Bingham, 1982). The fresh moringa leaves were used for determination of total soluble
proteins (Bradford, 1976), enzymatic antioxidants catalase (CAT), peroxidase (POD)
(Chance and Maehly, 1955), superoxide dismutase (SOD) (Giannopolitis and Ries, 1977)
and nonenzymatic antioxidants such as total phenolics (Ainsworth and Gillespie, 2007) and
ascorbic acid (Yin et al., 2008) were also determined (See details in section 3.6.3.3 and
3.6.3.4). The detailed analysis report is mentioned in Table 3.1.

3.3. Preparation of MLE


For preparation of MLE, leaf extraction was performed according to Price (2007), by
grinding young shoots (leaves and tender branches) with a pinch of water (1 L / 10 kg fresh
material) in a locally fabricated extraction machine (see Plate 1). After sieving through

27

Table 3.1 Bio-chemical composition of moringa leaf


Name of nutrient element / enzymes

Moringa leaf
(3replicates) S.E

Total soluble protein

1.40 0.003

-1

(mg g )
Enzymatic
antioxidants
-1

-1

(IU min mg
protein)

Non-enzymatic
antioxidants

Super oxide dismutase


(SOD) EC number (1.15.1.1)

191.86 8.482

Peroxidase (POD) EC
number (1.11.1.7)

21.99 0.073

Catalase (CAT) EC number


(1.11.1.6)

7.09 0.045

Total phenolic contents

8.19 0.007

(mg g

-1

GAE)
-1

Ascorbic acid (m mole g )


Nitrogen (%)
Phosphorus (%)
Potassium (%)
Calcium (%)
Magnesium (%)

1.933 0.145
0.180 .021
2.187 0.104
2.433 0.088
0.012 0.002
38.333 0.667

-1

Zinc (mg kg )

-1

3.500 0.289

Copper (mg kg )
-1

Iron (mg kg )

0.36 0.001

544.000 2.082
-1

Manganese (mg kg )

49.667 1.764

Boron (mg kg )

21.333 1.202

-1

28

29

cheese cloth the extract was centrifuged for 15 min at 8000 g. Various dilutions MLE0,
MLE10, MLE20, MLE30 and MLE40 (diluted to 0, 10, 20, 30 and 40 times with water
respectively) of the extract were prepared with distilled water then used in experiments for
priming as well as foliar spray.

3.4. Plant material


Seeds of wheat cv. Sehar-2006, tomato cv. Sahil and pea cv. Climax used as plant material
were obtained from Vegetable Research Institute and Wheat Research Institute of Ayub
Agriculture Research Institute, Faisalabad, Pakistan.

3.5. Crop season


The experiments were done during October-April 2008-2009 for pea and tomato and
November- April 2008-2009 and 2009-2010 for wheat crop. Fig. 3.1 and 3.2 represented the
meteorological data recorded during course of experiments.

3.6. Experimental sites


The analysis and optimization of MLE dilutions were done at laboratory, Dept. of Crop
Physiology, Univ. of Agriculture, Faisalabad. The assessment of method for exogenous
application of MLE was conducted in pea and tomato crop under green house and walk in
plastic tunnel (high tunnel), respectively. The evaluation of MLE as priming agent in wheat
was performed in pots under greenhouse conditions. The role of MLE in mitigating stress
like salinity and drought in wheat was assessed in pots under wire-net house conditions of
Department of Crop Physiology, University of Agriculture Faisalabad, Pakistan. The effect
of MLE on wheat late sowing stress was evaluated under field conditions of the
Experimental Farm of Department of Crop Physiology, University of Agriculture Faisalabad,
Pakistan during 2008- 10.

3.7. Experiment I: Optimization of moringa oleifera leaf extract (MLE)


dilutions
Design

Completely Randomized Design (CRD)

Replications 3
Medium

Petriplates (5 seeds in each plate)

30

Temperature(C) Max
R.Humidity(%)

Temperature(C) Min
Rain fall (mm)

80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
00.0
Oct-08

Nov-08

Dec-08

Jan-09

Feb-09

Mar-09

Apr-09

Fig. 3.1. Weather data for October 2008 to April 2009


Temperature(C) Max
R.Humidity(%)

Temperature(C) Min
Rain fall (mm)

100.0
80.0
60.0
40.0
20.0
0.0
Oct-09

Nov-09

Dec-09

Jan-10

Feb-10

Mar-10

Apr-10

Fig. 3.2. Weather data for October 2009 to April 2010


Source: Agricultural Meteorology Cell, Department of Crop Physiology, University
of Agriculture, Faisalabad

31

The detail of treatments was as under


i.

Control (Distilled water)

ii.

MLE20 (20 times diluted MLE)

iii.

MLE30 (30 times diluted MLE)

iv.

MLE40 (40 times diluted MLE)

The treatments were applied to completely moisten the Whatman no.1 filter paper. Seven
days after germination; seedlings were carefully harvested to record the data for the
following parameters.
3.7.1. Shoot and root lengths (cm)
Shoot and root lengths in centimeters were measured with the help of scale at the time of
harvest. The length was measured from the point where the root and shoot joins to the end of
root for root length and to the top of shoot for shoot length.
3.7.2. Shoot and root fresh and dry weights (g)
After harvesting the seedling, the shoot was cut from root at the point where they joined
together. The fresh weight was recorded for each part separately. The samples were dried in
an oven at 70C up to constant dry weight.
On the basis of higher germination and seedling vigor, 30 times diluted MLE was selected
for further studies.

3.8. Experiment II: Evaluation of MLE as priming agent


Completely Randomized Design (CRD)

Design

Replications 3
Medium

Earthen pots filled with 10 kg soil @ sand + silt + compost (1+1+1)


(see the soil analysis report presented in Table 3.2)

25 seeds per pot were sown. Three seedlings per pot were maintained at 10-days after sowing
(DAS). Tap water was used for irrigation when required. Fertilizer application was done @
120-100 kg NP ha-1.
Treatments
i.

Control (unprimed)

ii.

MLE10

iii.

MLE30

iv.

CaCl2 (-1.25 MPa)

(Farooq et al., 2006)

32

Table 3.2 Analysis report for soil filled in pots

Soil Characteristics

Results

ECe

4.03 dS m-1

pHs

7.53

SAR

12.65

TSS

45.06 m.mol L-1

Na+

33.14 m.mol L-1

Cl-

22.97 m.mol L-1

K+

0.47 m.mol L-1

Ece= Electrical conductivity, TSS= Total soluble salts, SAR= Sodium adsorption ratio

33

v.

HP (Hydro priming)

(Harris et al., 2001)

vi.

OFP (On-farm priming)

(Harris et al., 2001)

The priming period was evaluated according to (Harris et al., 2001). For CaCl2 priming,
seeds were soaked in aerated 50 mmol solution of CaCl2 for 12 h at 20C in the dark and
redried up to original weight with forced air under shade following Basra et al. (2005). In
hydropriming, the seeds were soaked in distilled water (1:5 seed to solution volume used)
and dried back near to original moisture with forced air under shade. 25 seeds were used in
each pot. After emergence, three seedlings were maintained per pot. Data for the following
parameters were collected.

3.8.1. Emergence parameters


3.8.1.1. Emergence index (EI)
EI was calculated according to the Association of Official Seed Analyst (1990):
EI= (number of germinated seeds / days of first count) +....+ (number of germinated
seeds / days of final count)
3.8.1.2. Mean emergence time (MET)
MET was calculated as described by Ellis and Robert (1981) as:
MET= ( Dn / n)
Where n is the number of seeds which were germinating on day and D is the number of days
counted from the beginning of emergence.
3.8.1.3. Time to 50% emergence (E50)
Time taken to 50 % emergence of seedlings was calculated according to Farooq et al. (2005):
N / 2 ni
E50 = ti +
(tj ti )
nj ni
Where N is the final number of germinated seeds; while ni and nj are the cumulative number
of seeds emerged by adjacent counts at the times ti and tj where ni < N/2 < nj.
After 14 days of emergence the seedlings will be evaluated for seedling vigor.

3.8.2. Seedling vigor:


3.8.2.1. Shoot and root lengths (cm)
Shoot and root lengths were measured as described in section 3.7.1.
3.8.2.2. Shoot and root fresh and dry weights (g)
Shoot and root fresh and dry weights were determined according to the section 3.7.2.
34

3.8.2.3. Leaf area plant-1 (cm2)


Leaf area was measured on leaf area meter (CI-203 Laser leaf area meter, CID Inc., USA) 75
days after sowing (DAS) when plants were fully grown.

3.8.3. Analytical parameters:


The leaves were analysed for following parameters.
3.8.3.1. Leaf chlorophyll contents
For determination of leaf chlorophyll contents, grinding of 0.5 g leaf sample was done in 80
% acetone to extract chlorophyll. The absorbance of filtrate was determined at 663 and 645
nm and the chlorophyll contents were calculated with the following formula described by
Arnon (1949).
Chlorophyll a (mg g-1) = [(0.0127 x A663 0.00269 x A645) x 100]/ 0.5
Chlorophyll b (mg g-1) = [(0.0229 x A645 0.00468 x A663) x 100]/ 0.5

3.8.3.2. Total soluble protein (mg g-1)


The determination of total soluble protein was consist of following steps
3.8.3.2.1. Total soluble protein extraction / Sample preparation
For extraction of total soluble proteins, 0.5 g of fresh plant material (leaves) was ground in a
pre-chilled pastor mortar by adding 1 mL extraction buffer with pH 7.2. Before extraction of
proteins cocktail protease inhibitors in a concentration of 1 M were added to the buffer. The
buffer used was phosphate buffer saline (PBS) containing 10 mM Na2 HPO4, 2 mM KH2 PO4,
2.7 mM KCl and 1.37 mM NaCl dissolved in distilled water and made up to a volume 1 L.
The pH 7.2 of PBS was adjusted with HCl and then autoclaved (Sambrook and Russell,
2001). The ground leaf material was centrifuged at 12000 x g for 5 min. Supernatant was
preserved in a separate centrifuge tube for the analysis of soluble proteins, while the pellet
was discarded.
3.8.3.2.2 Determination of total soluble protein
Total soluble proteins were determined followed by Bradford assay (Bradford, 1976). For
construction of standard curve 10, 20, 30, 40 and 50 g mL-1 were prepared from bovine
serum albumin (BSA) by adding 400 L Dye stock (Biorad, USA) and distilled water
followed by vortexed and incubation at room temperature for 30 min. The absorbance was
recorded at 595 nm using spectrophotometer (UV 4000 UV-VIS spectrophotometer). The
absorbance for the sample supernatant was also determined in a similar way. Concentration
35

(mg mL-1) of total soluble or heat stable fractions of proteins was determined from standard
curve (Fig. 3.3) prepared from absorbance of bovine serum albumin (BSA) and computed by
applying the formula: slope x absorbance / mL of extract used.

3.8.3.3. Enzymatic antioxidants (IU min-1 mg protein-1)


For enzymatic antioxidants determination of leaf sample, extraction was done in 5 ml of 50
mM phosphate buffer (pH 7.8), after centrifugation at 15000 g for 20 min, the supernatant
was

used

in

further

assay

for

superoxide

dismutase

(SOD)

activity

(Giannopolitis and Ries, 1977), peroxidase and catalase activity (Chance and Maehly, 1955)
by recoding absorbance at 560, 240 and 470 nm respectively.
3.8.3.3.1. Superoxide dismutase EC number (1.15.1.1) (IU min-1mg protein-1)
The SOD activity inhibits the photochemical reduction of nitroblue tetrazolium (NBT) at 560
nm. The monitoring of this inhibition is used to assay SOD activity. The reaction mixture
was prepared by taking 50 L enzyme extract and adding 1 mL NBT (50 M), 500 L
methionine (13 mM), 1mL riboflavin (1.3 M), 950 L (50 mM) phosphate buffer and 500
L EDTA (75 mM). This reaction was started by keeping reaction solution under 30 W
fluorescent lamp illuminations and turning the fluorescent lamp on. The reaction stopped
when the lamp turned off 5 min later. The NBT photo reduction produced blue formazane
which was used to measure the increase in absorbance at 560 nm. The same reaction
mixtures without enzyme extract in dark were used as blank. The SOD activity was
determined and expressed as SOD IU min-1 mg-1 protein (Giannopolitis and Ries, 1977).
3.8.3.3.2. Peroxidase (POD) EC number (1.11.1.7) (m.mol min-1 mg protein-1)
The POD activity assayed by guaiacol oxidation and defined as 0.01 absorbance change min1

mg-1 protein. The reaction mixture was prepared by adding 400 L guaiacol (20 mM), 500

L H2O2 (40 mM) and 2 mL phosphate (50 mM) in 100 L enzyme extract. The change in
absorbance at 470 nm of the reaction mixture was observed every 20 s up to 5 min. The POD
activity expressed as m.mol min-1 mg protein-1 (Chance and Maehly, 1955).

36

Table 3.3 Dilutions of bovine serum albumin (BSA)


BSA (L)

Water (L)

Dye stock (L)

Total volume (mL)

1600

400

10

1590

400

20

1580

400

30

1570

400

40

1560

400

50

1550

400

2
y = 0.0043x + 0.0906
2

R = 0.9924
0.35
0.30

Absorbance

0.25
0.20
0.15
0.10
0.05
0.00
0

Fig. 3.3.

10
20
30
Conc. of BSA standards

Standard curve of protein estimation

37

40

50

60

3.8.3.3.3 Catalase (CAT) EC number (1.11.1.6) ( mol min-1 mg protein-1)


The CAT activity assayed by the decomposition of H2O2 and change in absorbance due to
H2O2 was observed every 30 s for 5 min at 240 nm using a UV- visible spectrophotometer.
Reaction mixture for CAT contained 900 L H2O2 (5.9 mM) and 2 mL phosphate buffer (50
mM). Reaction was started by adding 100 L enzyme extract to the reaction mixture. The
Catalase activity was expressed as mol of H2O2 min-1 mg protein-1 (Chance and Maehly,
1955).

3.8.3.4. Non-enzymatic antioxidants


3.8.3.4.1. Total phenolic content (mg g-1)
Total phenols of leaf samples were determined at 760 nm using gallic acid as reference
standard (Ainsworth and Gillespie, 2007). To create calibration curve (Fig. 3.4) the standard
solutions were prepared in different concentrations i.e. 500, 250, 150 and 100 mg L-1 gallic
acid. For extraction and preparation of sample, 0.5 g leaf sample was homogenized in 5 mL
acetone (80 %). Filtration followed by extraction and volume of filtrate was raised with
acetone up to 10 mL. Then reaction mixture was prepared by taking 20 L sample or
standard and adding 1.58 mL water, 100 L Folin-Ciocalteu reagents within 30 s up to 8 min.
Reaction mixture was left at 40 oC for 30 min or at 25 oC for 120 minutes after adding and
mixing 300 L sodium carbonate. Absorbance of reaction mixture was read at 760 nm
against the blank (80% acetone). Total phenol levels in samples were determined by plotting
calibration curve from standards (Fig. 3.3) and reported as Gallic Acid Equivalent, GAE.
3.8.3.4.2 Ascorbic acid (m.mol g-1)
Ascorbic acid of leaf samples was measured according to Yin et al. (2008). The protocol is
given below:
3.8.3.4.2.1 Reagents
1. Trichloroacetic acid (TCA) 10%
2. Trichloroacetic acid (TCA) 5%
3. Phosphoric acid (44%)
4. 100 mM Phosphate Buffer Saline (BPS) PH 7.8
5. FeCl3 (3%)

38

Table 3.4 Preparation of Gallic acid standards


Stock solution
Gallic acid (g)

Ethanol (ml)

0.5

10

Water (ml)

Total volume (ml)

90

100

-1

Standard (mg L )
Stock (conc.) mg
mL-1

Stock (ml)

Water (ml)

Total volume (ml)

500
250
150
100

10
5
3
2

90
95
97
98

100
100
100
100

Fig. 3.4: Standard curve of total phenolic content estimation

39

6. 2,2- biphenyl (dissolved in 75% ethanol)


3.8.3.4.2.2. Standards
Standard curve was plotted using ascorbic acid as a reference standard. The stock solution of
100 g mL-1 or 10 mg 100mL-1 was prepared in distilled water. From stock solution different
dilutions i.e. 20, 40, 60, 80 and 100 g /mL were prepared (Table 3.5).
3.8.3.4.2.3. Procedure
0.5 g of plant material (leaf) was ground in a pre-cold mortar on ice in presence of 2 ml TCA
(5 %) with a little clean sand. After grinding, centrifuge at 12,000 x g for 20 min at 4oC. The
supernatant was used for ascorbate determination whereas pellet was discarded. Sample
mixtures was prepared by taking 200 L of the supernatant + 1.4 mL PBS and mixed well. It
was then incubated at room temperature for 1 min. After incubation, 0.4 mL TCA (10%),
44% phosphoric acid (0.4 mL), 0.2 mL 2, 2-biphenyl and 0.2 mL FeCl3 (3 %) were added
and mixed. This mixture was again incubated at 35 oC for 60 min in water bath. The
absorbance was measured at 525 nm using 100 mM PBS as a control. From this absorbance
value, ascorbic acid of samples (m mol g-1 fresh weight) was determined on the base of
standard curve plotted using absorbance value recorded for various standards (Fig.3.5).

3.8.4 Yield parameters


At maturity plants were harvested and threshed manually to record following yield related
parameters.
3.8.4.1 Number of grains per spike
The grains threshed and counted separately from spikes of each pot and then averaged for
number of grains per spike.
3.8.4.2 100 Grain weight (g)
From each pot, hundred grains were randomly separated and their weight was recorded.
3.8.4.3 Grain yield per plant (g)
Whole of the manually threshed produce were weighed to obtain grain yield per pot.

3.9. Experiment III: Evaluating the growth and development of Tomato to


exogenous application of MLE (pot study)
Design:

Factorial Completely Randomized Design (CRD Factorial arrangement)

Medium Earthen pots filled with 10 kg soil @ sand + silt + compost (1+1+1) (see the
soil analysis in Table 3.1)
40

Table 3.5 Dilutions for ascorbic acid standard solutions


Stock solution
(100g ml-1)

water (ml)

Total volume (g ml-1)

2 ml

20

4 ml

40

6 ml

60

8 ml

80

10 ml

100

1.40

y = -0.0139x + 1.4641
R = 0.9995

1.20

Absorbance 525nm

1.00
Absorbance

0.80

Linear
(Absorbance)

0.60

0.40

0.20

0.00
0

50

100

Connc.
Conc. of ascorbic
ascorbicacid
acidstandards
standards

Fig. 3.5 Standard curve of ascorbic acid estimation

41

150

Factor A (Modes of application):


1. Soil application;

2. Foliar spray

Factor B Treatments
i.

Control (distilled water)

ii.

MLE0

iii.

MLE10

iv.

MLE20

v.

MLE30

vi.

50 mg L-1 BAP

MLE foliar or soil applications were started 30 days after sowing @ 25 mL /plant and
repeated on weekly basis till plant maturity. Data for following parameters were recorded.
3.9.1. Number of vegetative branches per plant
Number of vegetative branches per plant was counted weekly and then was averaged.
3.9.2. Number of reproductive branches per plant
Number of reproductive branches per plant was counted weekly and then was averaged.
3.9.3 Number of flowers per plant
Number of flowers per plant were counted weekly and then averaged.
3.9.4. Number of fruits per plant
Number of fruits per plant were counted weekly and then averaged.
3.9.5. Fruit yield per plant (g)
The fruits harvested from individual plant and their weight was averaged
3.9.6. Leaf chlorophyll a and b contents (mg g-1)
Chlorophyll contents were calculated with the formula described by Arnon (1949) and
mentioned in section 3.8.3.1.
3.9.7. Total soluble proteins (mg g-1)
Total soluble proteins were determined by Bradford (1976) as described in section 3.8.3.2.
3.9.8. Enzymatic antioxidants (IU min-1 mg protein-1)
For determination of enzymatic antioxidants (IU min-1 mg protein-1) extraction and sample
preparation was done as described in section 3.8.3.3.

42

3.9.8.1. Superoxide dismutase (SOD) (IU min-1mg protein-1)


SOD activity was determined by Giannopolitis and Ries (1977) as described in section
3.8.3.3.1.
3.9.8.2. Peroxidase (POD) (m.mol min-1 mg protein-1)
POD activity was determined by following protocol of Chance and Maehly (1955) as
explained in section 3.8.3.3.2.
3.9.8.3. Catalase (CAT) ( mol min-1 mg protein-1)
Catalase activity was determined according to Chance and Maehly (1955) as given in section
3.8.3.3.3.
3.9.8.4. Leaf total phenolic contents (mg GAE g-1)
Total phenolic contents in leaf samples were determined as mentioned in section 3.8.3.4.1.
(Ainsworth and Gillespie, 2007).
3.9.8.5. Fruit lycopene contents (g g-1)
For determination of fruit lycopene contents, 1g fruit sample was ground / homogenized in
acetone-hexane (4:6) according to the method described by Fish et al. (2002). The sample
was shaked for 15 min on magnetic stirrer. Then 3 ml deionized water was added in each
sample. The sample was again shaked on ice for 5 min. The separation of sample in two
phases was achieved by leaving it at room temperature for 5 min. The absorbance of the
hexane (upper) layer was observed at 503 nm and hexane alone used as a blank (RaveloPrez et al., 2008). The lycopene contents were determined by using formula
Lycopene content (g g-1) = (A503 0.0007) 30.3/g tissue

3.10. Experiment IV: Evaluating the growth and development of pea to


application of MLE (pot study)
Design:

Factorial Completely Randomized Design (CRD Factorial arrangement)

Medium: Earthen pots filled with 10 kg soil @ sand + silt + compost (1+1+1) (see the
soil analysis in Table 3.5)
Factor A (Modes of application):
1. Soil application;

2. Foliar spray

Factor B Treatments
i.

Control (distilled water)

ii.

MLE0 (100 % pure MLE)


43

iii.

MLE10

iv.

MLE20

v.

MLE30

vi.

50 mg L-1 BAP

MLE foliar or soil applications were started 30 days after sowing @ 25 ml /plant and
repeated on weekly basis till maturity. Data for following parameters were collected.
3.10.1. Number of vegetative branches per plant
Same as given in section 3.9.1.
3.10.2. Number of reproductive branches per plant
Same as given in section 3.9.2.
3.10.3. Number of flowers per plant
Same as given in section 3.9.3.
3.10.4. Number of fruits per plant
Number of fruits per plant were counted weekly and then averaged.
3.10.5. Fruit yield per plant (g)
All the fruits harvested from an individual plant weighed and then averaged.
3.10.6. Leaf chlorophyll a and b contents (mg g-1)
Chlorophyll contents were calculated with the formula described by Arnon (1949) and
mentioned in section 3.8.3.1.
3.10.7. Leaf total phenolic contents (mg GAE g-1)
Total phenolic contents in leaf samples were determined as mentioned in section 3.8.3.4.1.
(Ainsworth and Gillespie, 2007).

3.11. Experiment V: Evaluating the response of late sown wheat to foliar


application of MLE under field conditions
Design

Randomized complete block design (RCBD)

Replications 3
Medium

Field conditions (Soil was clayey loam in texture)

Net plot size 5.0 m 2.0 m


Seed rate

110 kg ha-1

Sowing date 16 December, 2008

44

For all experiments of stress conditions, 30 times diluted MLE foliar spray was selected,
because it was found to be most effective in III and IV experiments conducted under normal
conditions.
Treatments:
T1 = Foliar spray of water (control)
T2 = Foliar application of MLE at tillering stage (t)
T3 = Foliar application of MLE at t + jointing stage (j)
T4 = Foliar application of MLE at t + j + booting stage (b)
T5 = Foliar application of MLE at t + j+ b+ heading stage
T6 = Foliar application of MLE at heading stage
The quantity of MLE used in foliar spray was decided after calibration of hand sprayer on the
basis of leaf saturation. All cultural practices were kept constant. Following parameters were
studied during course of experiment.

3.11.1. Growth Traits


3.11.1.1. Leaf area index (LAI)
Leaf area per plant in a unit area of 1 m2 was measured as explained in section 3.6.2.3. The
ratio of leaf area to ground area was estimated to determine leaf area index. Data on LAI
were started to record 50 DAS and continued on weekly bases up to 95 DAS.
3.11.1.2. Seasonal leaf area duration (SLAD) days
SLAD == (LAI1 + LAI2) x (T2 T1) / 2 (Reddy, 2004)
Where LAI1 was the leaf area index value recorded first time in the crop growing season and
LAI2 was the value of leaf area index calculated last time at crop maturity. T2 T1 was the
time interval of these two readings.
3.11.1.3. Crop growth rate (CGR) (g m-2 day-1)
For estimation of crop growth rate plants were harvested in a unit area of one m2 and oven
dried to obtain constant dry weight. This was started 50 DAS and continued up to 95 DAS on
weekly bases. The 4 values of CGR were calculated during this period by using the formula
mentioned below:
CGR = (W2 W1) / (T2 T1)

(Reddy, 2004)

W1 = oven dried weight at first sampling


W2 = oven dried weight at second sampling
45

T1 = time of first sampling


T2 = time of second sampling
3.11.1.4 Net assimilation rate (NAR) (g m-2 day-1)
Net assimilation rate was started to estimate from 50 DAS and repeated on weekly bases up
to 95 DAS. The 4 values of NAR were obtained during this period. Following formula was
used to determine NAR.
NAR

TDM/LAD

TDM

Total dry matter accumulated (W2 - W1)

LAD

(LAI1 + LAI2) x (T2 T1) / 2)

Where

3.11.2. Yield Traits


3.11.2.1. Number of fertile tillers (m-2)
At maturity, number of fertile tillers was counted manually in an area of 1 m2.
3.11.2.2. Number of grains per spike
At maturity, 10 spikes were selected randomly and their number of grains were counted and
then averaged.
3.11.2.3. 1000 grain weight (g)
After threshing, 1000 grains were randomly counted and weighed from the produce of each
plot.
3.11.2.4. Biological yield (t ha-1)
Whole above-ground plant biomass was harvested from each plot to measure biological yield
in kg, which was converted in to biological yield t ha-1.
3.11.2.5. Grain yield (t ha-1)
The produce of each plot threshed separately to obtain grain yield and then converted in to t
ha-1.
3.11.2.6. Harvest index (%)
The ratio of grain yield to biological yield was determined to obtain harvest index.

46

3.12. Experiment VI: Mitigating effects of salinity stress in wheat by MLE


application
30 times diluted MLE used as priming or foliar application and was compared with control, a
synthetic cytokinin (BAP) and H2O2 to evaluate the effectiveness of MLE in mitigating
salinity stress (Wahid et al., 2007).
Design

CRD (Factorial arrangement)

Replications

Medium:

soil+ compost + sand (1+1+1)

Seed rate:

25 seeds in each pot (At completion of emergence three


seedlings were maintained per pot)

Factor A Salinity levels (4, 8 and 12 dS m-1).


Prior to the development of salinity in soil medium used to fill the pots, analysis of soil was
carried out to determine the soil nutrients status (For Soil analysis, see Table 3.5). NaCl was
used to achieve the required level of salinity i.e. 4, 8 and 12 dS m-1 according to USDA
Laboratory Staff (1954). The soil was analysed again to confirm the actual level of salinity
developed.
Factor B

Treatments
i.

Control;

ii.

MLE30 foliar application

iii.

Benzyl amino purine foliar application (BAP, 50 mg L-1) (Amin et al., 2007)

iv.

H2O2 foliar application (120 M) (Wahid et al., 2007)

Foliar application was started one week after completion of emergence and kept repeating on
weekly basis @ 25 ml / plant up to maturity.

3.12.1 Seedling vigor


3.12.1.1. Shoot and root lengths (cm)
Shoot and root lengths were measured as described in section 3.7.1.
3.12.1.2. Shoot and root fresh and dry weights (g)
Shoot and root fresh and dry weights were determined as described in section 3.7.2.
3.12.1.3. Leaf area plant-1 (cm2)
Leaf area was measured as mentioned in section 3.8.2.3.
47

3.12.2. Analytical parameters


Leaves were randomly selected from each treatment after 75 days of sowing and analysed for
following parameters.
3.12.2.1 Leaf chlorophyll a and b contents (mg g-1)
Chlorophyll contents were calculated with the formula described by Arnon (1949) and
mentioned in section 3.8.3.1.
3.12.2.2. Leaf Na+ and K+ contents
The Na+ and K+ contents in leaves were determined by flame photometer (USDA Laboratory
Staff, 1954).
3.12.2.3. Leaf Cl- contents
The hot water treatment was used for estimation of leaf Cl- contents (USDA Laboratory Staff,
1954).
3.12.2.4. Total soluble protein (mg g-1)
Total soluble proteins were determined by Bradford (1976) as described in section 3.8.3.2.
3.12.2.5. Enzymatic antioxidants (IU min-1 mg protein-1)
For determination of enzymatic antioxidants (IU min-1 mg protein-1), the same protocol was
followed as described in section 3.8.3.3.
3.12.2.5.2. Superoxide dismutase (SOD) (IU min-1mg protein-1)
SOD activity was determined by Giannopolitis and Ries (1977) as described in section 3.8.3.3.1.

48

3.12.2.5.3. Peroxidase (POD) (m.mol min-1 mg protein-1)


POD activity was determined by following the protocol of Chance and Maehly (1955) as
explained in section 3.8.3.3.2.
3.12.2.5.4. Catalase (CAT) ( mol min-1 mg protein-1)
Catalase activity was determined according to Chance and Maehly (1955) as given in section
3.8.3.3.3.
3.12.2.6. Non enzymatic antioxidants
3.12.2.6.1. Total phenolic contents (TPC) mg g-1
Total phenolic contents in leaf samples were determined as mentioned in section 3.8.3.4.1.
(Ainsworth and Gillespie, 2007).
3.12.2.6.2. Ascorbic acid (mmole g-1)
Ascorbic acid of leaf samples was measured according to Yin et al. (2008) as given in
section 3.8.3.4.2.

3.12.3. Yield parameters:


At maturity plants were harvested and threshed manually to record following yield related
parameters.
3.12.3.1. Number of grains per spike
The procedure mentioned in section 3.6.4.1 was followed to estimate number of grains per
spike.
3.12.3.2. 100 Grain weight (g)
100 grain weight was determined as described in section 3.6.4.2.

3.13. Experiment VII: Mitigating effects of drought stress in wheat by


MLE application
Design

Factorial Completely Randomized Design (Factorial CRD)

Replications

Medium

Soil+ compost + sand (1+1+1) in earthen pots (soil analysis


given in Table 3.5)

Stage of stress induction:


Factor A

1 week old plants

Drought Levels
i.

Well watered

(100 % Field capacity)

ii.

Moderate drought

(75 % Field capacity)


49

iii.
Factor B

Severe drought

(50% Field capacity)

Treatments
i. Control
ii. MLE foliar application (optimized dilution level i.e. 30 times MLE)
iii. Benzyl amino purine foliar application (BAP, 50 mg L-1) (Amin et al., 2007)
iv. K+ (SOP 2%) (Ismail, 2005)

Foliar application was started one week after imposition of stress and repeated on weekly
basis @ 25 mL plant-1 up to maturity.
During course of experiment, the following observations were recorded.
3.13.1 Leaf area plant-1 (cm2)
Leaf area was measured as mentioned in section 3.8.2.3.

3.13.2 Analytical parameters


Leaves were randomly selected from each treatment 75 days after sowing (DAS) and
analysed for following parameters.
3.13.2.1 Leaf chlorophyll a and b contents (mg g-1)
Chlorophyll contents were analysed as mentioned in section 3.8.3.1. Chlorophyll contents
were calculated with the formula described by Arnon (1949).
3.13.2.2 Leaf Cl- contents
The hot water treatment was used for estimation of leaf Cl- contents following the protocol of
USDA Laboratory Staff (1954).
3.13.2.3. Total soluble protein (mg g-1)
Total soluble proteins were determined by Bradford (1976) as described in section 3.8.3.2.
3.13.2.4. Enzymatic antioxidants (IU min-1 mg protein-1)
For determination of enzymatic antioxidants (IU min-1 mg protein-1), extraction and sample
preparation was done as described in section 3.8.3.3.
3.13.2.4.1. Superoxide dismutase (SOD) (IU min-1mg protein-1)
SOD activity was determined by following the protocol of Giannopolitis and Ries (1977) as
described in section 3.8.3.3.1.
3.13.2.4.2. Peroxidase (POD) (mmol min-1 mg protein-1)
POD activity was determined by following protocol of Chance and Maehly (1955) as
explained in section 3.8.3.3.2.

50

3.13.2.4.3. Catalase (CAT) ( mol min-1 mg protein-1)


Catalase activity was determined according to Chance and Maehly (1955) as given in section
3.8.3.3.3.
3.13.2.5. Non enzymatic antioxidants
3.13.2.5.1. Total phenolic contents (TPC) (mg g-1)
Total phenolic contents in leaf samples were determined as mentioned in section 3.8.3.4.1. (Ainsworth
and Gillespie, 2007).
3.13.2.5.2. Ascorbic acid (m. mole g-1)
Ascorbic acid of leaf samples was measured according to Yin et al. (2008) as given in section 3.8.3.4.2.
3.13.2.6. Leaf K+ contents
The K+ content in leaves were determined by flame photometer (USDA Laboratory Staff, 1954).
3.13.3. Grain yield per plant (g)
At maturity plants were harvested and threshed manually to record grain yield per plant.

3.14. Statistical analysis


All the data collected for above-mentioned parameters in all experiments were statistically analysed by
determining significance of variance (P<0.05) using the MSTAT Computer Program (MSTAT
Development Team, 1989). Differences among means were compared by using Duncans New
Multiple Range test (Steel and Torrie, 1997).

51

Chapter 4
Result and Discussion
4.1 Experiment 1. Optimization of MLE dilution
In this experiment, moringa leaf extract (MLE) was diluted to 10, 20 and 30 times (MLE10,
MLE20 and MLE30) and applied to germinating wheat seeds to optimize MLE diluted dose.
Response was evaluated on the basis of seedling vigor. Exogenous application of varying
dilutions of MLE significantly enhanced the seed germination of wheat (Table 4.1.1, 4.1.2).
Among dilutions MLE30 was the most effective owing to maximum enhancement in seed
germination. MLE30 also induced earliness in germination as depicted by less mean time to
complete germination and time to 50% germination as compared to control (Fig. 4.1.1 b and
c). Likewise, exogenous application of MLE significantly enhanced shoots and roots fresh
and dry weights. Maximum enhancement in these growth attributes of wheat seedlings was
observed by MLE30 treatment (0.229 and 0.035 g; 0.201 g and 0.058 g, respectively). In the
same way, shoot and root length of wheat seedlings increased by addition of MLE in the
growth medium. However, maximum enhancement in both shoot and root length was
observed when MLE30 was applied (Fig. 4.1.2). Nonetheless, 30 times diluted MLE was the
most effective in enhancing wheat seedling vigour in present studies. On the basis of these
findings MLE 30 was selected for further experiments in pot and field conditions.

Discussion
Plant extracts of some trees and crop residues have been reported to influence crop growth
and yield (Guenzi and McCalla, 1962; Chung and Miller, 1995; El-Atta and Bashir, 1999;
Ahmed and Nimer, 2002; Farooq et al., 2008). Leaf extract of M. oleifera is one such
example. It has been reported that foliar application of M. oleifera leaf extract accelerated
growth of tomato, peanut, corn and wheat at early vegetative growth stage, improve
resistance to pests and diseases and enhanced more and larger fruits and generally increase
yield by 20 to 35% (Fuglie, 2000). In our studies, the exogenously applied MLE also
effectively improved seed germination and seedling vigour as compared to untreated control.
However, improving effect of MLE on growth was concentration dependent. Of various
dilutions, exogenous application of 30 times dilution was the most effective in improving

52

growth of wheat seedlings as has earlier been observed in cotton and sugarcane (Foidle et al.,
2001). While Fuglie (2000) reported yield enhancement by 25-30% in onions, bell pepper,
soya, maize, sorghum, coffee, tea, chili, melon by MLE application. He suggested that this
growth and yield enhancement was due to presence of zeatin, a cytokinin in moringa leaves.
In another study with sorghum, maize and wheat, Phiri (2010) found that spray with 10 times
diluted MLE to germinating seeds of these cereals increased germination of sorghum, length
of maize radicals and hypocotyls of wheat. However, seed germination of wheat reduced
significantly due to spray of 10 times dilution of MLE. In the present study, 30 time dilution
was more effective in germination and seedling growth of wheat than other dilutions. So, it is
suggested that MLE has potential to enhance the seed germination, and seedling growth in
wheat. However, effectiveness of exogenous application of MLE depends on type of species,
dilution of MLE and plant developmental stage. In view of published reports, it is suggested
that Moringa leaf extract posses a rich and rare combination of nutrients, amino acids,
antioxidants and cytokinin as observed in the present study (Table 3.1), and exogenous use of
MLE might have enhanced plants endogenous hormonal levels thereby resulting in
increased seed germination and seedling growth.

53

Table 4.1.1 Mean sum of squares of data for germination index (GI), mean germination
time (MGT), and time to 50% germination (T50) of wheat grown in petri plates treated
with Moringa oleifera leaf extract (MLE).
SOV

df

Germination index
(GI)

Mean germination
time (MGT)

Time to 50%
germination (T50)

Treatments

14.572*

0.262*

2.814*

Error

6.396

0.349

0.838

Table 4.1.2 Mean sum of squares of data for final germination percentage, shoot fresh
weight and shoot dry weight of wheat seedling grown in petri plates treated with MLE.
SOV

df

Final germination
percentage

Shoot fresh weight

Shoot dry weight

Treatments

100.100**

0.0022**

0.0003**

Error

88.978

0.00004

0.00003

Table 4.1.3 Mean sum of squares of data for shoot length, root fresh weight and root
dry weight of wheat seedling grown in petri plates treated with MLE.
SOV

df

Shoot length

Root fresh weight

Root dry weight

Treatments

3.1724**

0.0143**

0.00093**

Error

0.00004

0.0001

0.00001

**P<0.01

*P<0.05

54

Table 4.1.4 Mean sum of squares of data for root length of wheat seedling grown in
petri plates treated with MLE.
SOV

df

Root length

--

--

Treatments

1.0849**

--

--

Error

0.00020

--

--

**P < 0.01

55

LSD 0.05p= 1.684

(a)

LSD 0.05p= 0.6094

14.00

3.50
a

Time to 50% germination (T50)

8.00
6.00
4.00
2.00
0.00

bc
2.00

1.50
1.00
0.50

Dilutions of MLE

(d)
a

LE
40
M

LE
30

Co
nt
ro
l

LE
40
M

LE
30
M

LE
20

102.00
100.00
98.00
96.00
94.00
92.00
90.00
88.00
86.00
84.00

LE
20

ab

ab

LSD 0.05p= 3.299

Final germination percentage

(b)

Co
nt
ro
l

Mean germination time (MGT)

2.50

0.00

LSD 0.05p= 0.3932


3.50
3.40
3.30
3.20
3.10
3.00
2.90
2.80
2.70

3.00

Germination Index (GI)

12.00
10.00

(c)

Dilutions of MLE

Fig4.1.1. Effect of different dilutions of Moringa oleifera leaf extract (MLE)

on germination index (a), mean germination time (MGT) (b), time to


50% germination (T50) (c) and final germination percentage of
wheat cv. Sehar-2006 under laboratory conditions.

56

(a)

shoot fresh weight LSD 0.05p= 0.004


shoot dry weight LSD 0.05p= 0.003

0.300
a

Weight (g)

0.250

0.200

0.150
0.100

a
b

0.050

0.000
root fresh weight LSD 0.05p= 0.006

( b)

root dry weight LSD 0.05p= 0.002

0.300

Weight (g)

0.250
a

0.200
0.150

0.100
a

d
0.050

0.000
(c)

shoot length LSD0.05=0.012


root length LSD0.05= 0.027

a
bc

LE
40
M

LE
30

LE
20

Co
nt
ro
l

Length (cm)

10.00
9.00
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00

Dilutions of MLE
Fig.4.1.2. Effect of different dilutions of Moringa oleifera leaf extract (MLE) on shoot

fresh and dry weight (a), root fresh and dry weight (b) and shoot, root length
(c) of wheat cv. Sehar-2006 under laboratory conditions.

57

4.2. Experiment II: Evaluation of MLE as priming agent


In this experiment, effects of optimized dose of MLE i.e., MLE30 as seed priming agent was
compared with other priming agents (Hydropriming, CaCl2, on-farm priming) and
emergence, seedling vigour, antioxidants status and yield of wheat were assessed. The results
show that seed priming with different priming agents significantly increased the emergence
index and time to 50% emergence (T50) of wheat seed (Table 4.2.1). Maximum seed
emergence index was obtained in seeds primed with MLE30 followed by CaCl2 and
hydropriming. The on-farm priming remained at bottom and statistically at par with control
for emergence index (Fig. 4.2.1a).
Analysis of variance (ANOVA) of the data for mean time taken by the seeds to emerge show
that seed priming with different priming agents did not affect mean time to emerge (Fig.
4.2.1b). In contrast, time to 50% emergence (T50) showed that seed priming significantly
reduced time to attain T50. However, maximum reduction in time to 50% emergence in wheat
was observed in MLE30 (Fig.4.2.1c).
Seed priming showed a variable but significant stimulatory effect on seedling vigor attributes
(Table 4.2.2 and Table 4.2.3). The maximum improvement in shoot fresh and dry weight was
observed in seedlings raised from seeds primed with MLE30 followed by MLE10. All of the
other priming treatments under study were statistically different from each other but better
than control. Minimum shoot fresh and dry weight was obtained in control (Fig. 4.2.2 a, b).
Similarly significantly highest root fresh and dry weight was produced when seeds were
primed with MLE30 (Fig.4.2.2 d,e) followed by hydropriming and MLE10. MLE10 seed
priming ranked third regarding root fresh weight.
The significantly highest increment in shoot length was found with CaCl2 seed priming
followed by MLE30 seed priming (Fig. 4.2.2 c). The shoot length was observed decreased in
order of hydropriming > on- farm priming > MLE10 priming > control.
MLE30 and MLE10 primed seeds outperformed regarding root length, however, MLE30 was
the superior one (Fig. 4.2.2 f). The root length observed in case of CaCl2 shorter than MLE
priming but longer than hydropriming and on farm priming. The maximally reduced root
length was found in non primed seed.
The largest leaf area was produced in seedlings emerged from MLE30 primed seeds followed
by MLE10 (Fig. 4.2.3 a). The hydropriming and on-farm priming showed significantly

58

similar values for leaf area. While comparing priming treatments CaCl2 produced minimum
value for leaf area, although, it was lesser than other priming treatments still larger than
control. Overall MLE30 produced comparatively more vigorous seedlings.
Priming also showed significant influence on leaf chlorophyll contents (Table 4.2.4) which
was maximally increased by MLE30 followed by CaCl2 seed prirning (Fig. 4.2.3b). The
MLE10 exhibited chlorophyll a content statistically lesser than MLE30 and CaCl2 but higher
than on-farm priming and hydropriming. Nevertheless the on-farm priming and
hydropriming were significantly better for chlorophyll a contents as compared to control.
The lowest recorded value of chlorophyll a was found in leaves of plants raised from non
primed seeds.
Similar to chlorophyll a the significantly highest quantities of chlorophyll b were also
obtained in case of MLE30 primed seeds (Fig. 4.2.3c). The CaCl2 and hydropriming were
statistically similar although inferior to MLE30 but superior than other priming treatments.
The performance of non primed seeds was better than on-farm priming. The more
pronounced effects of seed priming were observed with MLE30 and CaCl2 for leaf
chlorophyll a and b contents.
In case of yield attributes effects of priming treatments were found significant (Table 4.2.5).
More and heavier grains were obtained from MLE30 primed plants which resulted in highest
grain yield per plant. All the priming treatments gave more yield than non primed control
(Fig. 4.2.4 a, b and c)
Total leaf soluble protein contents showed positive response towards seed priming treatments
(Table 4.2.6). MLE30 gained significantly highest protein contents whereas MLE10
significantly lesser than MLE30 but higher than other treatments (Fig. 4.2.5a). Hydropriming
exhibited better leaf protein contents than CaCl2 and on-farm priming. The protein contents
obtained in CaCl2 and on-farm priming were even lesser than control.
More enhancement in super oxide dismutase (SOD), peroxidase (POD) and catalase (CAT)
were resulted from MLE30 seed priming agent as compared to other priming and non primed
seed treatments. The hydropriming followed the MLE30 in case of SOD (Fig. 4.2.4 b) and
POD but in case of CAT MLE30 was followed by MLE10 seed treatment (Fig. 4.2.5 c and
d). The least activity of SOD, CAT and POD was observed when seeds were on-farm primed

59

and primed with CaCl2, respectively. Nevertheless, the seed enhancement techniques
significantly affect the enzymatic antioxidants.
A significant increase in ascorbic acid was also found in case of MLE10 seed priming
followed by MLE30 (Fig. 4.2.5 e). The plants raised from hydro primed seed produced
ascorbic acid lesser than moringa priming but higher than CaCl2 and on- farm priming.
Although least ascorbic acid but maximum total phenolic content were produced in CaCl2
priming treatments (Fig. 4.2.5 f).

Discussion
In this study, effects of different priming agents on growth and yield of wheat were
evaluated. From the results of the present study, it is obvious that seed priming enhanced
speed and total final germination count of wheat. Maximum increase in seed emergence and
speed of seed germination was observed in plants raised from MLE30 treatment (Table 4.2.1,
Fig. 4.2.1 a, b, c). It has already reported that 30 times diluted moringa leaf extract
significantly increased seed and seedling vigor in wheat (Afzal et al., 2008), maize (Basra et
al., 2011) and range grasses i.e. Cenchrus ciliaris, Panicum antidotale and Echinochloa
crusgalli (Nouman et al., 2011) reported that seed priming with MLE30 significantly
increased germination of all three range grasses. All seed priming agents enhanced the
seedling shoot and root fresh and dry weight of wheat plants, particularly when seeds were
primed with MLE30. However, seed priming with CaCl2 caused maximum increase in shoot
length while longer roots were observed with MLE30 priming (Fig. 4.2.2 c and f). Seed
priming enhances rate of metabolism which results in increase in speed of germination and
emergence (Ashraf et al., 2008). The effectiveness of moringa is because its leaves are rich
source of PGR hormone, zeatin, ascorbic acid, Ca and K (Fuglie, 1999; Foidle et al., 2001),
which are involved in modifying the seed germination and seedling establishment related
metabolism.
Osmohardening with CaCl2 was previously successfully used to improve emergence and
plant height in rice (Farooq et al., 2006). So, its effectiveness was also confirmed in wheat by
present studies. It might be due to the fact that higher or enhanced mobilizations of
metabolites/inorganic solutes to germinating plumule result in enhanced growth (Taiz and
Zeiger, 2002).

60

The crop yield is ultimate goal for cereal cultivation and numbers of strategies are underway
to increase the productivity. Crop yield in cereals is mainly determined by optimum plant
population, number of grains and size of grains. A number of reports are available which
show that seed priming enhanced the crop productivity by either increasing the emergence,
number of grains or size of grain or by combination of these traits (Khan et al., 2006; Ashraf
et al., 2008; Athar et al., 2008). Farooq et al. (2006) reported that highest value of 1000
kernel weight in rice was produced by KCl followed by CaCl2 seed priming. In the present
study, seed priming with different priming agents increased the number of grains spike-1 and
100 grain weight. Better partitioning of photoassimilates in developing grains at grain filling
stage causes increase in size of grain (Taiz and Zeiger, 2002). The level of cytokinin is
reported positively correlate to final grain weight in maize (Dietrich et al., 1995) and since
moringa leaves are rich in zeatin, a cytokinin (Foidle et al., 2001) so, MLE priming
effectiveness might be due to its other growth promoting factors. Gupta et al. (2003) also
reported that benzyl adenine, a cytokinin, could be used to improve sink and source capacity
of wheat in increasing grain yield. In some earlier studies it has been observed that seed
priming with plant growth regulators, inorganic salts, compatible solutes or sugar beet extract
improved seed germination by providing physiological and biochemical adaptations (Pill and
Savage, 2008; Afzal et al., 2006a). Similarly, priming with cytokinins like kinetin or benzyl
amino purine (BAP) increased salt tolerance in wheat at seedling stage (Iqbal and Ashraf,
2006).
The enhanced yield by seed priming arise from the events taking place during earlier stages
of crop growth such as faster production of more vigorous seedlings (Farooq et al., 2006).
Ruan et al. (2002) also observed an improvement in seedling vigor with CaCl2 and CaCl2 +
NaCl priming in greenhouse conditions.
Seed priming with MLE30 followed by CaCl2 also improved the photosynthetic pigments.
CaCl2 priming is well known priming tool for rice (Farooq et al., 2006), we tried in present
study for wheat and found effective in enhancing the speed and spread of emergence and
seedling vigor as well, though not as affective as MLE30 results. The vigorous seedling as
exhibited by more chlorophyll in leaves. Afzal et al. (2011) also found CaCl2 as effective
priming agent in tomato.

61

In the present study, seed priming with different priming agents caused an enhancement in
leaf total soluble protein. However, this effect was more in MLE30 priming. While working
with wheat Al-Hakimi and Hamada (2001) observed that seed priming with ascorbic acid
increased leaf soluble proteins. According to Price (2000) moringa leaf extract contain
ascorbic acid in appreciable quantities. Thus, increased leaf protein due to MLE30 seed
priming was one of the reasons that contributed in improved growth of wheat. Similarly,
remarkable increase in protein content was obtained from CaCl2 seed priming under salinity
(Afzal et al., 2006b) which may be due to better defence of membrane and membrane bound
enzymes.
It is evident that priming with antioxidant compounds such as ascorbic acid and tocopherol
can increase free radical scavenging enzymes such as superoxide dismutase (SOD), catalase
(CAT) and peroxidase in seeds (Chang and Sung, 1998). Moringa leaves are richest source of
antioxidants. It is reported that about 46 antioxidants are present in moringa leaves. The
major ones are ascorbate, carotenoids, phenols and flavonoid (Iqbal and Bhanger, 2006). So
the pretreatment of seeds with MLE improved the total phenolic contents of maize seedlings
(Basra et al., 2011). In the present study, it was observed that most of priming treatments
were effective in not only improving seedling vigor and may be attributed to the
counteraction of free radicals and synthesis of membrane-bound enzymes as in other nonprimed seeds.
Seed priming improved the seedling vigor and increased the activity of scavenging enzymes
in leaves of wheat and as indicated by increase in SOD and POD by MLE30, CAT and TPC
by CaCl2 and ascorbic acid by MLE10 seed priming (Fig. 4.2.5 b, c, d, e and f). Catalase,
which is involved in the degradation of H2O2 into water and oxygen, is the major H2O2
scavenging enzyme in all aerobic organisms. Previous reports show that priming resulted in a
great enhancement in CAT and SOD activities in plants (Basra et al., 2004). The
hydropriming decreased CAT activity in the present study which confirms the findings of
Srinivasan and Saxena (2001) who reported that CAT activity was not increased after
hydropriming in radish. Therefore, it is likely that enhanced antioxidant enzyme activity in
wheat cultivars due to MLE30 and CaCl2 priming was due to highest contents of antioxidants
found in MLE (Iqbal and Bhanger, 2006).

62

Table 4.2.1 Mean sum of squares of data for emergence index (EI), mean emergence
time (MET), time to 50% emergence (E50) of wheat raised from seeds primed with
different priming agents.
SOV
Priming
Treatments
Error

df

Emergence index
(EI)

Mean emergence
time (MET)

Time to 50 %
emergence (E50)

3.100*

0.629ns

0.282**

12

0.985

0.646

0.016

Table 4.2.2 Mean sum of squares of data for shoot fresh weight, shoot dry weight and
shoot length of wheat raised from seeds primed with different priming agents.
SOV
Priming
Treatments
Error

df

Shoot fresh weight

Shoot dry weight

Shoot length

0.070**

0.088**

7.324**

12

0.001

0.001

0.032

Table 4.2.3 Mean sum of squares of data for root fresh weight, root dry weight and
root length of wheat raised from seeds primed with different priming agents.
SOV
Priming
Treatments
Error
**P< 0.01

df

Root fresh weight

Root dry weight

Root length

0.128**

0.045**

16.902**

12

0.001

0.001

0.163

*P<0.05

ns Non significant

63

Table 4.2.4 Mean sum of squares of data for leaf area, leaf chlorophyll a and
chlorophyll b contents of wheat raised from seeds primed with different agents.
SOV
Priming
Treatments
Error

df

Leaf area

Chlorophyll a

Chlorophyll b

932.835**

0.605**

0.304**

12

7.238

0.001

0.001

Table 4.2.5 Mean sum of squares of data for number of grains per spike, 100 grain
weight and grain yield per plant of wheat raised from seeds primed with different
priming agents.
SOV
Priming
Treatments
Error

df

Number of grains
per spike

100 grain weight

Grain yield per plant

353.428**

0.147**

0.870**

12

0.039

0.001

0.001

Table 4.2.6 Mean sum of squares of data for total soluble protein, and enzymatic
antioxidants i.e. superoxide dismutase (SOD) and peroxidase (POD) of wheat raised
from seeds primed with different agents.
SOV
Priming
Treatments
Error

df

Total soluble
protein

Super oxide
dismutase (SOD)

Peroxidase
(POD)

0.090**

160.045**

56.769**

12

0.001

0.160

0.011

** p < 0.01

64

Table 4.2.7 Mean sum of squares of data for enzymatic antioxidant catalase (CAT)
and non-enzymatic antioxidants i.e. total phenolic content and ascorbic acid of wheat
raised from seeds primed with different agents.
SOV
Priming
Treatments
Error

df

Catalase (CAT)

Total phenolic
content

Ascorbic acid

117.126**

1.100**

0.0009**

0.001

0.001

12

0.007

**P< 0.01

65

(a)

LSD 0.05p = 1.766

Emergence Index (EI)

15

a
ab

abc

12

ab

bc

9
6
3
0
(b)

LSD 0.05p = ns

Mean emergence time (MET)(days)

20

15

10

0
(c)
b

H
P

30

O
FP

Ca
Cl
2

M
LE

8
7
6
5
4
3
2
1
0

co
tro
l
M
LE
10

Time to 50% Emergence E50 (days)

LSD 0.05p = 0.226

Priming treatments
Fig 4.2.1. Effect of seed priming on emergence index (EI) (a), mean

emergence time (MET) (b) and time to 50% emergence (E50) (c) of
wheat cv. Sehar-2006. (MLE10 =10 times diluted MLE, MLE30=
30 times diluted MLE, HP=Hydro priming, OFP= On-farm
priming)

66

(a)

LSD 0.05p = 0.004

1.5
c

f
1.0

0.5

Root fresh weight (g)

Shoot fresh weight (g)

1.5

(d)

LSD 0.05p= 0.005

0.0

1.0

c
0.5

0.0
(b)

LSD 0.05p = 0.006

(e)

LSD 0.05p= 0.003


1.00

1.00
b

0.75

c
0.50

0.25

Root dry weight (g)

0.75

0.50

a
b
c

0.25

0.00

0.00

(c)

LSD 0.05p = 0.316

25.0

15.0
10.0

5.0

Roor length (cm)

20.0

(f)

LSD 0.05p = 0.719

25.0

20.0

a
c

15.0
10.0
5.0

Priming treatments

Fig4.2.2. Effect of seed priming on shoot fresh weight (a), shoot dry weight

(b), shoot length (c), root fresh weight (d), root dry weight (e) and
root length (f) of wheat cv. Sehar-2006. (MLE10= 10 times diluted
MLE, MLE30= 30 times diluted MLE, HP=Hydro priming, OFP=
On farm priming)

67

P
OF

FP
O

P
H

2
Ca
Cl

LE
30
M

LE
10
M

nt
ro
l
Co

Priming treatments

Ca
Cl
2

0.0

0.0

Co
nt
ro
l
M
LE
10
M
LE
30

Shoot length (cm)

HP

Shoot dry weight (g)

(a)

LSD 0.05p = 4.785

Leaf area (cm2)

100

80
60

40
20
0
(b)

LSD 0.05p= 0.027

3.5

-1

Chlorophyll a (mg g fw)

4.0
b

3.0

2.5
2.0
1.5
1.0
0.5
0.0

(c)

OF
P

Ca
Cl
2

LE
30

LE
10

Co
n

HP

4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
tro
l

-1

Chlorophyll b (mg g fw)

LSD 0.05p= 0.031

Priming treatments
Fig.4.2.3. Effect of seed priming on leaf area (a), chlorophyll a (b) and

chlorophyll b (c) of wheat cv. Sehar-2006. (MLE10= 10 times


diluted MLE, MLE30= 30 times diluted MLE, HP=Hydro priming,
OFP= On farm priming)

68

(a)

LSD= 0.05p 0.353

Number of grains spike

-1

60

a
b

50
c
40
30

d
e

20
10
0

(b)

LSD 0.05p= 0.048


3.5

100 Grain weight (g)

2.5

3.0

b
c

2.0
1.5
1.0
0.5
0.0
(c)

LSD 0.05p= 0.045

(g)
-1

Grain yield plant

3.50

3.00

2.50
2.00

1.50
1.00
0.50

O
FP

H
P

Ca
Cl
2

0
M
LE
3

Co
nt
ro
l
M
LE
10

0.00

Priming treatments
Fig.4.2.4. Effect of seed priming on number of grains per spike (a), 100 grain

weight (b) and grain yield per plant (c) of wheat cv. Sehar-2006
(MLE10= 10 times diluted MLE, MLE30= 30 times diluted MLE,
HP=Hydro priming, OFP= On-farm priming)

69

a
b

(b)

-1

15
c
d
e

10
5

(c)

b
c

25
d

20

15
10
5
0

3.0

-1

1.0
0.5

1.5

Priming treatments

1.0
0.5
0.0
Co
n

O
FP

H
P

Co
nt
ro
l
M
LE
10
M
LE
30
Ca
Cl
2

0.0

2.0

Priming treatments

Fig.4.2.5. Effect of seed priming on leaf total soluble protein (a), enzymatic
antioxidants (superoxide dismutase, peroxidase and catalase) (b,d,c) and
non enzymatic antioxidants (ascorbic acid and total phenolic contents) (e,
f) of wheat cv. Sehar-2006. (MLE10= 10 times diluted MLE, MLE30= 30
times diluted MLE, HP=Hydro priming, OFP= On farm priming)

70

FP

l
M
LE
10
M
LE
30

2.5

tro

1.5

Total phenolics (mg GAE g )

2.0
-1

(f)

LSD 0.05p= 0.043

(e)

LSD 0.05p= 0.002

Ascorbic acid (m mole g )

b
15

Ca
Cl
2

10

20

30

-1

-1

20

25

LSD 0.05p= 0.225

30

-1

Superoxide dismutase (IU mg min protein)

LSD 0.05p = 0.711

25

30

Peroxidase (IU mg min protein)

Total soluble proteins (mg g-1 fw)

2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0

(d)

LSD 0.05p= 0.51

Catalase (IU mg-1min-1 protein)

(a)

LSD 0.05p= 0.014

4.3. Experiment III:


Evaluating the growth and development of tomato to exogenous application of MLE
(pot study)
In a preliminary research study tomato plants were treated with different dilutions of (MLE0,
MLE10, MLE20, and MLE30) and BAP (50 mg L-1). The exogenous application was done
either through soil or foliar. The data collected for different growth, yield and antioxidants
status are discussed below:
Data presented in Table 4.3.1 and Fig. 4.3.1a elucidate that exogenous application of MLE
and BAP significantly affected the number of vegetative branches per plant. The foliar
application of BAP along with soil and foliar application of MLE30 showed maximum
enhancement in number of vegetative branches. The soil application of control was superior
to foliar application of control, MLE0 and MLE10. The minimum number of vegetative
branches was observed where plants were sprayed with undiluted MLE.
Foliar application of BAP, MLE30 and soil application of MLE20 exhibited higher number
of flowering branches (Table 4.3.1a and Fig. 4.3.1. b). Regarding control, MLE0 and MLE10
soil application showed more flowering branches as compared to their foliar application. The
lowest number of flowering branches was found in case of foliar applied MLE0 and MLE10.
The significant differences were found for number of flowers plant-1 (Table 4.3.1) with
largest number of flowers obtained in foliar spray of MLE30 alone (Fig.4.3.2a). Although the
number of flowering branches obtained in MLE30 was lesser than BAP foliar spray,
however, more number of flowers per branch was obtained in MLE30 than BAP.
The more flowering branches in foliar application of BAP and more number of flowers in
foliar applied MLE30 resulted in highest number of fruits in both the treatments (Fig. 4.3.2
b). The lesser number of flowers in foliar spray of MLE10, MLE0 and control lead to the
least number of fruits in these treatments.
Data on the response of fresh weight of fruit to the exogenous treatments indicate that the
foliar application of MLE30 not only exhibited large number of flowers and more number of
fruits but also heaviest fruits were observed in the same treatment (Fig.4.3.2c). Although the
same number of flowers were produced by foliar application of BAP and MLE30 but lighter
weight fruits in BAP as compared to MLE30 were obtained. The minimum fruit weight was
recorded in case of foliar applied MLE10, MLE0 and control.

71

Analysis of variance (ANOVA) of the data in Table 4.3.2 and 4.3.3 depict that exogenous
application significantly affected leaf chlorophyll a and chlorophyll b contents. The foliar
application of BAP maximally raised chlorophyll a contents followed by foliar application of
MLE30 (Fig. 4.3.3 a, b). In case of all exogenous applications irrespective of their
application method more chlorophyll a contents were observed as compared to control. In
contrast, the highest chlorophyll b contents were found in case of foliar spray of water.
Data presented in Table 4.3.3 and Fig. 4.3.4a showed that maximum leaf total soluble
proteins were obtained by foliar application of BAP, MLE30 and MLE20. Non significant
difference between soil and foliar application was found in case of MLE10 and control. The
lowest quantity of total soluble protein was obtained under foliar application of MLE0.
The exogenous applications of different concentrations of MLE significantly affected the
antioxidant activities of tomato leaves (Table 4.3.4).The MLE30 foliar application ranked
first in case of enzymatic antioxidants i.e. SOD, POD and CAT (Fig. 4.3.4 b, c, d). Other
treatments such as foliar applied BAP and MLE20 exhibited SOD value statistically at par
with MLE30. The least quantity of SOD was obtained under foliar application of MLE0. The
foliar applied BAP and MLE20 produced statistically similar POD value which was lesser
than foliar applied MLE30. The POD lowest value was found when plants received foliar
spray of water (control).
Nevertheless, the quantity of CAT observed in tomato leaves was lesser than POD but higher
than SOD. The foliar applied MLE30 was superior for CAT and its soil application showed
CAT lower than MLE foliar but better than other treatments under study. All the treatments
performed better than control for CAT contents. Similar trend were observed in case of leaf
total phenolics and fruit lycopene contents in which foliar application of MLE30
outperformed all the growth enhancers under study (Fig. 4.3.4 c and f ).
Overall, foliar mode of exogenous application and MLE30 dilution were more effective
regarding growth, yield and biochemical parameters of tomato.

Discussion
The exogenous application of growth promoting compounds i.e. PGR, antioxidants, vitamins,
minerals, osmoprotectants etc. are being used to enhance the crop growth and economic yield
(Adams and Adams, 2002; Al-Hakimi and Hamada, 2001; Azeem and Ahmad, 2011).
Researchers are always interested to find natural compounds containing these growth

72

promoting factors as cheap, natural and environmentally friendly compounds. Foliar


application of yeast, a natural source of cytokinin, sugar, vitamins, amino acid and proteins in
appreciable quantities increased growth, chlorophyll contents and green pod yield of
Phaseolus vulgaris (El-Tohamy and El- Greadly, 2007). Azeem and Ahmad (2011) observed
that both individual and combined foliar application of K, Fe and B caused the significant
improvement in number of fruits, weight of fruits, leaf chlorophyll and protein contents of
tomato. We tried to use moringa leaf extract as a natural source of cytokinin, nutrients and
antioxidants. In present study the maximum vegetative growth was obtained under foliar
application of BAP, however, the yield related parameters such as number of flowering
branches, number of flowers, number of fruits and fruit weight per plant were higher by
foliar application of 30 times diluted moringa leaf extract (Fig. 4.3.1 and 4.3.2). It may be
attributed to the higher nutrient requirements of reproductive phase fulfilled by application of
macro and micro nutrient containing MLE. However, the response of plant to MLE depends
upon both plant growth stage (as more response was observed during reproductive growth)
and MLE concentration. The more diluted MLE spray provides nutrients to the plant through
stomata and enhances the yield. These results confirmed the findings of Wu and Lin (2000)
who found that higher concentration of seaweed extract resulted in sticky brown cotyledons
due to drop in biological activity of extract. According to Crouch and Staden (1991)
hormones at low concentration often promote physiological response while inhibit it at
higher concentration. But in case of soil application concentrated MLE performed better than
diluted MLE.
In our studies the plants treated with BAP and MLE in either method of exogenous
application showed a significant enhancement in photosynthetic pigment chlorophyll a as
compared to control but the maximum chlorophyll b was observed in case of soil application
of water (Fig.4.3.3). These findings are in accordance to Kumari et al. (2011) in which
largest content of chlorophyll a, b and carotenoids in tomato were obtained by foliar
application and soil drench + foliar application treatments. Previously, increased levels of
photosynthetic pigments in tomato leaves with application of Ascophyllum nodosum extract
(Whapham et al., 1993), and effective chlorophyll syntheses in maize and phaseolus mungo
by application of Sargassum seaweed extract (Lingakumar et al., 2004) was observed.

73

In earlier reports the high level of soluble protein in maize can be maintained by application
of 1% and 0.5% Sargassum seaweed extract (Lingakumar et al., 2004) and in sorghum with
1.5% liquid extract obtained from Hydroclathrus clathratus (Ashok et al., 2004). In present
research the highest protein content was achieved in plants where foliar application of
MLE30 was done (Fig. 4.3.4). This increase protein contents may be due to enhanced
availability and absorption of minerals which facilitated the source efficiency of leaves. The
use of diluted extract or lower concentration caused more increase in protein contents may be
due to enhanced absorption of necessary elements in such a concentration (Anantharaj and
Venkatesalu, 2001).
In the present research MLE and BAP significantly improved the enzymatic and
nonenzymatic antioxidants of tomato leaves depending upon their concentration and method
of application. Under the exogenous application of concentrated and diluted MLE and BAP,
the maximum activities of SOD, POD, CAT and total phenolics were obtained with foliar
application of MLE30.
Dorais et al. (2008) reported that the injurious effects e.g. age-related molecular
degeneration, cancer and cardiovascular disorders in humans caused by various substances
can be counteracting by improving lycopene contents. Due to its importance there is
increasing trend for developing lycopene rich food product and ingredient (Choudhary et al.,
2009). In our study foliar application of MLE30 produced highest lycopene contents while in
case of soil application MLE0 or higher concentration of MLE showed larger lycopene
contents as compared to control (Fig. 4.3.4f). Similarly highest contents of lycopene in
tomato fruit were produced by soil application of 10% seaweed extract (Kumari et al., 2011).
From such report it is evident that cytokinin like substances or cytokinin itself present in
aqueous extract of moringa leaf or Sargassum johnstonii extract facilitate mobilization of
nutrients to the fruit and improve lycopene contents.
Although it was a preliminary research study, however, the present research findings
strongly suggested that foliar application of MLE30 was most effective and has a potential to
be used as plant growth enhancer in tomato crop.

74

Table 4.3.1 Mean sum of squares of data for number of vegetative branches, number of
flowering branches and number of flowers of tomato under exogenous application of
different plant growth enhancers.
SOV

df

Number of
vegetative
branches plant-1

Number of
flowering
branches plant-1

Number of flowers
plant-1

16.333*

38.521*

218.180**

Treatment

41.683**

99.571**

1135.229**

Application
method x
Treatment

37.133**

46.271**

582.126**

Error

36

3.042

3.021

9.011

Application
method

Table 4.3.2 Mean sum of squares of data for number of fruits, fruit yield plant-1 and leaf
chlorophyll a of tomato under exogenous application of different plant growth
enhancers.
SOV

df

Number of
fruits plant-1

Fruit yield plant-1

Leaf chlorophyll a

164.628*

0.261*

0.440**

Treatment

703.421**

2.471**

0.797**

Application
method x
Treatment

346.885**

1.308**

0.197**

Error

36

10.589

0.019

0.001

Application
method

** p < 0.01

* p < 0.05 ns = non significant

75

Table 4.3.3 Mean sum of squares of data for leaf chlorophyll b, plant height and leaf
total soluble protein of tomato under exogenous application of different plant growth
enhancers.
SOV

df

Leaf
chlorophyll b

Plant height

Leaf total soluble


protein

0.266**

1.346**

0.266*

Treatment

0.505**

2.443**

0.505**

Application
method x
Treatment

0.551**

0.123**

0.551**

Error

36

0.002

0.003

0.002

Application
method

Table 4.3.4 Mean sum of squares of data for super oxide dismutase (SOD), peroxidase
(POD) and catalase (CAT) of tomato leaf under exogenous application of different plant
growth enhancers.
SOV

df

Leaf superoxide
dismutase (SOD)

Leaf peroxidase
(POD)

Leaf catalase (CAT)

0.063*

74.018**

0.314*

Treatment

0.128**

122.737**

2.168**

Application
method x
Treatment

0.028*

9.502**

0.198*

Error

36

0.014

0.016

0.314

Application
method

** p < 0.01

* p < 0.05

76

Table 4.3.5 Mean sum of squares of data for leaf total phenolic contents (TPC) and fruit
lycopene of tomato under exogenous application of different plant growth enhancers.
SOV
Application

df
1

Total Phenolic
contents (TPC)

Fruit lycopene

135.445**

--

9.882**
--

method

Treatment

41.857**

24.828**

Application
method x
Treatment

15.446**

7.914**

Error

36

3.143

0.023

** p < 0.01

77

--

---

(a)

LSD 0.05p= 4.305


Soil

Foliar
a
a

-1

Number of vegetative branches plant

30.0

bc

efg

25.0

ab
bc

fg

20.0

15.0
10.0
5.0
0.0
(b)

LSD 0.05p= 2.493


-1

Number of flowering branches plant

30.0
a

25.0
20.0

bc
d

cd

ab

ab
d

15.0

10.0
5.0
0.0
n
Co

tro

LE
M

LE
M

10

LE
M

20

LE
M

30

P
BA

Fig 4.3.1. Effect of exogenous application of growth enhancer on number of

flowering branches (a) and number of vegetative branches (b) of


tomato cv. Sahil (MLE0, MLE10, MLE20, MLE30 =0, 10, 20, 30
times diluted MLE respectively, BAP= benzyl amino purine)

78

(a)

LSD 0.05p= 4.305


Soil

Foliar

Number of flowers plant-1

120.0

a
b

100.0
d

de

80.0

f f

60.0
40.0
20.0
0.0
(b)

LSD 0.05p= 4.667


120.0
a

Number of fruits plant

-1

100.0
80.0
e e

bc b

bcd

bc

a
cd

60.0
40.0
20.0
0.0

(c)

LSD 0.05p= 0.1977


a

6.0

-1

Fruit yield plant (g)

5.0
4.0

de

f f

cd c

cd

de

3.0
2.0
1.0

BA
P

M
LE
30

M
LE
20

M
LE
10

0
M
LE

Co
nt
ro
l

0.0

Fig 4.3.2. Effect of exogenous application of growth enhancer on number of

flowers plant-1 (a), number of fruits (b) and fruit weight (c) of tomato
cv. Sahil (MLE0, MLE10, MLE20, MLE30 = 0, 10, 20,30 times
diluted MLE respectively, BAP= benzyl amino purine).

79

(a)

LSD 0.05p= 0.150

Total Soluble Protein (mg g-1)

7.0
6.0

ab
dd

Soil

Foliar

dd

bc a

5.0
4.0
3.0
2.0
1.0

POD (IU min-1 mg-1 protein)

Soil

(d)

LSD 0.05p= 0.181

0.0
(b)

LSD 0.05p= 0.170

100
90
80
70
60
50
40
30
20
10
0

Foliar

f e

gh

i h

cb

(e)

LSD 0.05p= 0.638


15

4.0

de e

de

abc
cde

ab
bcda

bcd

1.0
0.0

cd

a
bc

9
6
3

(f)

LSD 0.05p= 0.218


60
Fruit lycopene contents

50

gh
h

fg i

ec

a
d

40
30
20
10

nt
ro
l
M
LE
0
M
LE
10
M
LE
20
M
LE
30

0
Co

BA

Co

(c)

a
a
b
b bcd
cd cd bcd
bc
cd
d
cd

nt
ro
l
M
LE
0
M
LE
10
M
LE
20
M
LE
30

Leaf total phenolics

bcd
bc
bcd bc

LSD 0.05p= 0.078


50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0

12

2.0

de
de

de

cd

BA

3.0

Catalase (IU min-1 mg-1 protein)

SOD (IU min-1 mg-1 protein)

5.0

Fig 4.3.3. Effect of exogenous application of growth enhancer on total soluble

protein (a), SOD (b), total phenolics (c), POD (d), catalase (e) of leaf
and fruit lycopene (f) of tomato cv. Sahil (MLE0, MLE10, MLE20,
MLE30 = 0, 10, 20, 30 times diluted MLE respectively, BAP= benzyl
amino purine)

80

(a)

LSD 0.05p= 0.045


Soil

Foliar

Leaf chlorophyll a (mg g-1)

2.5
2.0
1.5

e
f

i h

1.0
0.5
0.0
(b)

LSD 0.05p= 0.064

Leaf chlorophyll b (mg g-1)

2.5
2.0
a

1.5

b
d

de

1.0
f

g
i

0.5
0.0

LSD 0.05p= 0.078

(a)

5.0
a

Plant height (m)

4.0
3.0

g e

f f

a
b

2.0
1.0

P
BA

LE
30
M

LE
20
M

LE
10
M

LE
0
M

Co

nt
ro
l

0.0

Fig 4.3.4. Effect of exogenous application of growth enhancer on chlorophyll a

(a), chlorophyll b (b) and plant height (c) of tomato cv. Sahil
(MLE0, MLE10, MLE20, MLE30 = 0, 10, 20, 30 times diluted MLE
respectively, BAP= benzyl amino purine)

81

4.4. Experiment IV:


Evaluating the growth and development of pea to exogenous application of
MLE (pot study)
The growth and yield response of pea under soil and foliar application of different growth
enhancers was studied. The results obtained are mentioned and discussed below:
The soil and foliar application of different growth enhancers significantly affect the
vegetative, reproductive and biochemical parameters of pea (Table 4.4.1, 4.4.2 and 4.4.3).
The foliar spray of MLE30 produced maximum number of vegetative branches which were
significantly superior to all other treatments under study (Fig. 4.4.1 a). The foliar spray of
BAP, MLE20 and soil application of MLE30 exhibit statistically similar vegetative branches
lesser than MLE30 but higher than control. More number of vegetative branches was
observed in foliar application as compared to soil application. Among the MLE dilutions and
BAP, the MLE30 performed best. The least number of vegetative branches similar to control
were obtained in case of soil applied MLE10 along with both foliar and soil applied MLE0.
The highest number of vegetative branches turned in to reproductive branches and produced
highest number of flowers when the plants received foliar spray of MLE30 (Fig. 4.4.1 b, c).
It was followed by the foliar spray of MLE20, BAP and soil applied MLE20. There was no
significant difference among soil applied MLE20, MLE10, MLE0 and control in case of
reproductive branches and number of flowers per plant.
The exogenous application in the form of soil and foliar spray significantly affect the number
of vegetative, reproductive branches and number of flowers. It leads to the highest number of
pods and pod yield (weight) under foliar application of MLE30 (Fig. 4.4.2).The soil
application did not perform better than foliar spray in any growth and yield determing
attributes.
Although the different growth enhancers significantly affect growth and yield, however, their
effect in case of chlorophyll a and b was non significant. All the growth enhancers effect leaf
chlorophyll contents in similar way, nevertheless largest chlorophyll a and b were obtained
when leaves were sprayed with MLE30.
While observing effect of soil and foliar applied growth enhancers on antioxidant status of
pea, highest quantities of total phenolic contents were found under foliar spray of BAP (Fig.
4.4.3c). It was statistically at par with soil applied 20 or 30 times diluted MLE. Unlike, other
82

growth and yield parameters more enhancements in total phenolic content were obtained by
the soil application as compared to foliar application. However, all soil and foliar treatments
were significantly better than control.

Discussion
Exogenous applications of plant extract are known to enhance crop growth and yield. Pea
crop yield like majority of grain legumes greatly depends on the number of flowering
branches, number of flowers, and number of pods per plant or per unit area. In present
research the exogenous application of nutrient rich MLE affected these traits significantly.
The application of BAP exhibited slight effect on the growth and yield attributes of pea as
compared to MLE. The plants that were sprayed with MLE30 showed 37 and 60% increase
in flowering branches and pod yield respectively, as compared to control. Accordingly,
Duval and Shetty (2001) reported that 1.5 % anise root extract (AR-10) showed enhancement
in pea growth. The probable reason may be the water soluble characteristics of cytokinin
found in AR-10 root tissues (Andarwulan and Shetty, 1999) similarly the zeatin a cytokinin
in MLE might be reason of 25-30% growth and yield enhancement in onions, bell pepper,
soya, maize, sorghum, coffee, tea, chili, melon (Fuglie, 2000).
The important attributes of a food product considered by a consumer are its physical
appearance and colour. The colour of foods depends upon the presence of various natural or
artificial pigments produced during growth or after harvest. The green color of vegetables is
due to chlorophyll whereas carotenoids and/or anthocyanins provide yellow, red and orange
colors to fruits and vegetables. All the organisms capable of carrying out photosynthesis
possess carotenoids and chlorophylls, however, the chlorophylls often masked the bright
colors of many carotenoids in photosynthetic tissues (Bartley and Scolnik, 1995). In green
peas chlorophylls a and b were identified as the major chlorophylls (Edelenbos et al., 2001).
In our study both method of exogenous application and concentration of growth enhancers
exhibit non significant difference in case of chlorophyll a and b (Fig. 4.4.3 a and b). It may
be attributed that MLE is a rich source of Fe and Mg (Nambiar, 2006). Both these elements
important for chlorophyll biosynthesis (Marschner, 1995). Mg as a component of chlorophyll
and Fe although not constituent of chlorophyll but involved in chlorophyll biosynthesis i.e. in
conversion of Mg proporphyrin in to chlorophyllide (Marschner, 1995). So enhanced
chlorophyll a and b constituent under application of Fe and Mg rich MLE may not be

83

affected by the concentration and method of application. Furthermore, the chlorophyll


contents in pea much influenced by genotype (Kidmose and Grevsen, 1992).
Polyphenols are important redox-active antioxidants found in high concentration in
vegetables and fruits (Odukoya et al., 2007). Phenolic compounds considered as powerful
chain-breaking antioxidants (Shahidi and Wanasundara, 1992) and their scavenging ability
based upon their hydroxyl groups (Hatano et al., 1989). In the present study, the leaf total
phenolic content of pea in foliar application of BAP was found to be highest i.e. 5.82 mg
GAE g-1 (Fig. 4.4.3) followed by soil applied MLE 30 (5.592 mg GAE g-1) as compared to
the control (3.152 mg GAE g-1). In previous studies the AR-10-yeast extract treatments
improved the phenolic content in pea seedlings as compared with the control (Duval and
Shetty, 2001).
From the above discussion it become clear that MLE as a blend of mineral nutrients,
antioxidants and PGR like cytokinin is an effective natural plant growth enhancer for
promotion of vegetative and reproductive growth and antioxidant phenols in pea.

84

Table 4.4.1 Mean sum of squares of data for number of vegetative branches, number of
reproductive branches and number of flowers of pea under exogenous application of
different plant growth enhancers.
SOV

df

Number of
vegetative
branches plant-1

Number of
reproductive
branches plant-1

Number of flowers
plant-1

30.250**

40.111**

0.111ns

Treatment

32.028**

54.067**

65.711**

Application
method x
Treatment

3.183*

5.178*

16.978**

Error

24

0.583

1.444

1.639

Application
method

Table 4.4.2 Mean sum of squares of data for number of pods, pods weight plant-1 and leaf
chlorophyll a of pea under exogenous application of different plant growth enhancers.
SOV

df

Number of
pods plant-1

Pods weight plant-1

leaf chlorophyll a

42.250**

9.020*

0.0001ns

Treatment

82.583**

114.518**

0.001**

Application
method x
Treatment

20.050**

7.027**

0.0001ns

Error

24

1.472

0.412

0.0001

Application
method

**P < 0.01


*P < 0.05
ns = non significant

85

Table 4.4.3 Mean sum of squares of data for leaf chlorophyll b and total phenolic
contents (TPC) of pea under exogenous application of different plant growth enhancers.
Leaf
chlorophyll b

0.001**

Total phenolic
contents (TPC)
3.361**

Treatment

0.0001*

3.686**

--

Application
method x
Treatment

0.0001ns

0.662**

--

Error

24

0.0001

0.049

--

SOV
Application

df

---

method

**P < 0.01


*P < 0.05
ns = non significant

86

(a)

LSD 0.05p= 1.28


Foliar application

Number of vegetative branches plant

-1

Soil application
20.0
15.0

a
10.0
5.0

bc bc
fg
de
efg
efg

ef
g

cd

de

0.0
(b)

Number of reproductive branches plant

-1

LSD 0.05p= 2.02

20.0

bc

bc
cd

de

15.0

fg

ef
efg

efg

10.0
5.0
0.0

(c)

LSD 0.05p= 1.082


a

25.0

Number of flowers plant

-1

bc
20.0
efg
ef

fg

g de
15.0

bc
cd

efg

g
10.0
5.0

BA
P

LE
30
M

LE
20
M

LE
10
M

LE
0
M

Co
nt
ro
l

0.0

Fig 4.4.1 Effect of exogenous application of different growth enhancers on

number of vegetative branches (a), reproductive branches (b) and


number of flowers per plant (c) of pea cv. Climax. (MLE0, MLE10,
MLE20, MLE30 = 0, 10, 20, 30 times diluted MLE respectively,
BAP= benzyl amino purine)

87

(a)

LSD 0.05p= 2.045

Number of pods plant

-1

Soil application
20.0
18.0
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0

Foliar application
a
b

b
c

c
de

cd

de

ef
f

LSD 0.05p = 1.082

(b)

20.0

18.0
b

-1

Pod yield plant (g)

16.0

12.0

d
e

10.0

ef
f

8.0

ef

6.0
4.0

b
c

14.0

2.0

Fig 4.4.2

BA
P

LE
30
M

LE
20
M

LE
10
M

LE
0
M

Co
nt
ro
l

0.0

Effect of exogenous application of different growth enhancers on


number of pods (a) and pod yield plant-1 (b) of pea cv. Climax.
(MLE0, MLE10, MLE20, MLE30 = 0, 10, 20,30 times diluted MLE
respectively, BAP= benzyl amino purine)

88

(a)

LSD 0.05p= 0.063 ns


Soil application

Foliar application

-1

Cholorophyll a (mg g fw)

0.20
0.15
0.10
0.05
0.00
(b)

LSD 0.05p= 0.063 ns

-1

Cholorophyll b (mg g fw)

0.20

0.15

0.10

0.05

0.00
(c)

7.0
6.0
5.0
4.0

Fig 4.4.3

bc
abc

de
f

ab
bcd

a
cde

3.0
2.0
1.0

BA
P

LE
30
M

LE
20
M

LE
10
M

LE
0

0.0
Co
nt
ro
l

-1

Total phenolics (mg GAE g )

LSD 0.05p= 0.0373

Effect of exogenous application of different growth enhancers on


chlorphyll a (a), b (b) and total phenolics (c) of pea cv. Climax.
(MLE0, MLE10, MLE20, MLE30 = 0, 10, 20, 30 times diluted MLE
respectively, BAP= benzyl amino purine)

89

4.5. Experiment V:
Response of late sown wheat to foliar application of MLE under field conditions
Foliar application of MLE at different growth stages revealed significant improvement in
growth, development and yield as compared to control (water spray) (Table, 4.5.1,4.5.2,
4.5.3, 4.5.4, 4.5.5, 4.5.6, 4.5.7). The crop attained the maximum leaf area index (LAI) 75
days after sowing (DAS) with the highest value of LAI under foliar applications of MLE at
all critical stages i.e. tillering, jointing, booting and heading (T + J + B + H). While MLE
application at other growth stages produced LAI although lesser than T + J + B + H while
check remained at bottom (Fig. 4.5.1). Nonetheless, a decreasing trend was observed in LAI
after 75 DAS but this reduction was minimal in plants with MLE applied at all growth stages
followed by foliar application at heading, while maximum reduction was observed in control
plants (Fig. 4.5.1). Effect of MLE application on seasonal leaf area duration (SLAD) was
also significant (P<0.05), and plants having MLE foliar application stayed green longer than
control (4.5.1). However, higher SLAD (65.4 d) were recorded in plants sprayed at all stages
closely followed by MLE spray at heading alone (62.63 d) than control (56.18 d) (Fig. 4.5.1).
Foliar spray of MLE caused a gradual rise in crop growth rate of late sown wheat crop and
showed maximum growth rate (9.74 g m-2day-1 ) in foliar spray at 4 stages (Fig. 4.5.2).
Afterwards, the growth rate of crop decreased but the minimum reduction (5.97 g m-2 day-1)
was observed in case of 4 MLE foliar sprays followed by CGR produced in MLE application
only at heading (5.47 g m-2 day-1) while the maximum reduction (4.41 g m-2 day-1) was in
untreated plants (Fig. 4.5.2). Maximal gain in net assimilation rate was observed up to 75
DAS as compared to control under MLE foliar spray at any growth stage with subsequent
reduction and the crop subjected to 4 foliar sprays showed least reduction (2.46 g m-2 day-1)
in net assimilation rate followed by NAR produced in MLE at heading (2.35 g m-2 day-1), the
highest reduction (2.20 g m-2 day-1) was exhibited by foliar spray of water (Fig. 4.5.2).
Similarly, response of MLE on yield and its related traits was significant. Maximum number
of fertile tillers and grains per spike, 1000-grain weight, biological, and economic yield and
harvest index were recorded when MLE was sprayed at tillering + jointing + booting +
heading crop stages as compared to control (Fig. 4.5.3 & 4.5.4). Nonetheless, similar number
of fertile tillers and grains per spike were recorded where MLE was applied at T+J and
T+J+B stages (Fig. 4.5.3). But the response in case of 1000-grain weight, biological and

90

economic yield as well harvest index varied among the foliar applications at different stages
and was highest in tillering + jointing + booting + heading spray followed by heading
treatment. However, there was no significant (P>0.05) difference for these traits when MLE
was applied at tillering, tillering + jointing and tillering + jointing and booting crop stages
(Fig. 4.5.3, 4.5.4), whilst minimum values for these traits were observed in plants with only
water application. Nevertheless, the performance of MLE at any growth stage was better than
control and the pronounced effects of MLE were observed when it was sprayed at four crop
stages (T + J + B + H) with the maximum contribution being observed under MLE
application at heading.
Discussion
Late sowing of wheat shortens the growth period a pre-requisite for harvesting higher yield
(Farooq et al., 2008). The postponement of wheat sowing after mid November induces yield
reduction by 50 kg ha-1 per day (Khan, 2004). Abrupt rise in temperature during early spring
further intricate the problem (Wardlaw and Wrigley, 1994). In cool season cereal species,
heat stress turns down the chlorophyll contents leading to many physiological damages; leaf
senescence the major one (Xu and Huang 2008). Spano et al. (2003) emphasized that when
assimilates supply to the grain decreased due to acceleration in senescence as in case of late
sown wheat then delaying leaf senescence may be an advantageous attribute. MLE foliar
spray at tillering, jointing, booting and heading sstages showed highest value of seasonal leaf
area duration (Fig. 4.5.1) due to delayed leaf senescence which resulted in 10.70% increment
in grain yield. It might be due to cytokinin in the MLE, which has stay green induction
quality as indicated by exogenously applied BAP enhanced the grain yield of Kalyansona
and HD 2285 cultivars of wheat by 8.8 and 13.70%, 5.66 and 13.33%, under normal and late
sown conditions, respectively (Gupta et al., 2003). When the transportation of cytokinins is
reduced to leaves under heat stress, it increases the degree of senescence (early maturity).
Cytokinin containing MLE application may negates the early maturing effects of heat stress
exhibiting longer leaf area duration. In addition, moringa leaf is also rich in ascorbate,
carotenoids, phenols, potassium and calcium like other plant growth enhancers (Foidle et al.,
2001). Antioxidants such as ascorbic acid and glutathione are found at high concentrations in
moringa chloroplasts and other cellular compartments are crucial for plant defense against
oxidative stress (Noctor and Foyer, 1998). Under combined heat and drought stress a rise in

91

wheat grain yield and more stable cell membrane and chlorophyll were observed by
exogenous application of cytokinin (Gupta et al., 2000). Thermotolerance and yield stability
in maize was reported by Cheikh and Jones (1994) as a result of high cytokinin content in
maize kernel for the heat stress period. Under late planting of wheat, maintenance of
photosynthetic activity due to increased temperatures during maturation (Paulsen, 1994) and
efficient utilization of these photosynthates indicated by high harvest index, (Gifford and
Thorne, 1984; Blum et al., 1994) are two important determinants of grain yield. The
maximum recorded harvest index (Fig. 4.5.4) by MLE sprayed at all stages supported that
photosynthetic activity was maintained up to maturity which was result of longer seasonal
leaf area duration and more stay green period. This delayed onset of leaf senescence is
reported to provoke about 11% more carbon fixation in Lolium temulentum (Thomas and
Howarth, 2000). An extension in active photosynthetic period may enhance total
photosynthates availability in annual crops life cycle and higher mass per grain can be
achieved if assimilated carbon supply be maintained to grain during grain filling period
(Spano et al., 2003).
The MLE application at single stage of heading although was statistically different but
closely followed the MLE treatment at four growth stages regarding 1000 grain weight
(40.85 g), biological yield (13.65 t ha-1), grain yield (3.19 t ha-1) and harvest index (23.39),
which may be due to the ability to maintain green leaf area duration stay green throughout
grain filling period, remobilization of soluble carbohydrates during grain filling (Stoy, 1965)
and by significant increment in grain weight of wheat by attracting more assimilates towards
the developing grain with the application of benzyl adenine at anthesis (Warrier et al., 1987).
In later phases of grain filling, leaf senescence caused shortage of assimilate then extended
duration of photosynthesis provides more photoassimilate translocated to the grain, improved
the grain weight as an outcome of amplified carbohydrate content.

92

Table 4.5.1 Mean sum of squares of data for LAI (50 DAS), LAI (57 DAS) and LAI (75
DAS) of wheat by exogenous application of MLE under late sown conditions.
SOV
Replication

df
2

Treatment

Error

10

LAI (50 DAS)

LAI (57 DAS)

LAI (75 DAS)

0.001

0.001

0.006

0.001*
0.000

0.009*
0.000

0.032*
0.003

Table 4.5.2 Mean sum of squares of data for LAI (85 DAS), LAI (95 DAS) and seasonal
leaf area duration of wheat by exogenous application of MLE under late sown
conditions.
SOV

df

LAI (85 DAS)

LAI (95 DAS)

Seasonal leaf area


duration

Replication

0.001

0.001

0.037

Treatment

0.102**

0.048**

Error

10

0.000

0.000

28.069**
0.155

Table 4.5.3 Mean sum of squares of data for CGR (57 DAS), CGR (75 DAS) and CGR
(85 DAS) of wheat by exogenous application of MLE under late sown conditions.
Replication

SOV

df
2

Treatment

Error

10

**P<0.01

*P<0.05

CGR (57 DAS)

CGR (75 DAS)

0.005

0.001

0.005

0.066*
0.022

0.621**
0.003

1.121**

LAI = Leaf area index


DAS = Days after sowing
CGR = Crop growth rate g m-2 day-1

93

CGR (85 DAS)

0.019

Table 4.5.4 Mean sum of squares of data for CGR (95 DAS), NAR (57 DAS) and NAR
(75 DAS) of wheat by exogenous application of MLE under late sown conditions.
Replication

SOV

df
2

CGR (95 DAS)

NAR (57 DAS)

NAR (75 DAS)

0.007

0.002

0.003

Treatment

0.874**

0.006

0.019*

Error

10

0.011

0.010

0.001

Table 4.5.5 Mean sum of squares of data for NAR (85 DAS), NAR (95 DAS) and
number of fertile tillers of wheat by exogenous application of MLE under late sown
conditions.
SOV

df

NAR (85 DAS)

NAR (95 DAS)

Number of fertile
tillers

Replication

0.001

0.001

0.703

Treatment

0.026*

0.021*

447.051**

Error

10

0.003

0.003

2.774

Table 4.5.6 Mean sum of squares of data for number of grains per spike, 1000 grain
weight and biological yield of wheat by exogenous application of MLE under late sown
conditions.
SOV

df

Number of
grains per
spike

1000 grain weight

Biological yield

Replication

0.222

0.002

0.001

Treatment

5.256*

5.345**

0.232**

Error

10

0.356

0.014

0.001

**P<0.01

*P<0.05

CGR = Crop growth rate g m-2 day-1


NAR = Net assimilation rate g m-2 day-1
DAS = Days after sowing

94

Table 4.5.7 Mean sum of square summaries of the data for grain yield and harvest index
of wheat crop by exogenous application of MLE under late sown conditions.
SOV

df

Grain yield

Harvest index

--

Replication

0.001

0.007

--

Treatment

0.033**

0.342**

--

Error

10

0.000

0.004

--

**P<0.01

*P<0.05

95

(a)

LSD 0.05p (50 DAS)= .019, LSD (57DAS)=0.019, LSD 0.05p (75DAS)=
0.099, LSD0.05p (85 DAS)=0.019, LSD 0.05p (95 DAS) = 0.019

Leaf area index

dist. Water
MLE foliar spray at t + Jointing(j)
MLE foliar spray at t +j+b+heading
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

MLE foliar spray at tillering(t)


MLE foliar spray at t+j+booting(b)
MLE foliar spray at heading

a a a b
b a
c
f e d

a b

c b a ab a c

e e d c

a b

b a a a a b

50

57

75

85

95

Days after sowing (DAS)


(b)

Seasonal leaf area duration (days)

LSD 0.05p= 0.7162


68
66
64
62
60
58
56
54
52
50

a
b
c

cd

d
e

r
ate
W
.
st
Di

til

t)
g(
ir n
le
t+

j)
g(
tin
n
i
Jo

j+
t+

b)
g(
in
t
o
bo
t+

j+

g
in
ad
e
h
b+

g
in
ad
e
h

Stages of MLE foliar spray


Fig. 4.5.1. Effect of exogenous application of MLE on leaf area index (a) and

seasonal leaf area duration (b) of wheat cv. Sehar-2006 under late
sown conditions.

96

(a)

LSD 0.05p (57 DAS)= 0.270, LSD0.05p (75 DAS)= 0.099


SD 0.05p (85) DAS= 0 .251, LSD 0.05p (95DAS)= 0.191
dist. Water
MLE foliar spray at t + Jointing(j)
MLE foliar spray at t +j+b+heading

MLE foliar spray at tillering(t)


MLE foliar spray at t+j+booting(b)
MLE foliar spray at heading

b a ab a

10.0

a
c
b

-2

-1

Crop growth rate (g m day )

12.0
a a a

8.0
a
6.0
4.0

e d c

b a a a a b

2.0
0.0
57

75

85

95

(b)

Net assimilation rate (g m-2 day-1)

LSD 0.05p (57 DAS)= ns, LSD 0.05p (75 DAS) = 0.575,
LSD 0.05p (85 DAS)= 0.099, LSD 0.05p (95 DAS) =0.099
4.00
a a a a a
b
c bc bc bc

3.00

a ab
c b b b

a b

2.00

1.00

0.00
57

75

85

95

Days after sowing (DAS)


Fig.4.5.2. Effect of exogenous application of MLE on crop growth rate (a) and

net assimilation rate (b) of wheat cv. Sehar-2006 under late sown
conditions.

97

(a)

LSD 0.05p= 3.030

-2

Number of fertile tillers (m )

390
a

380
370
b

360

350

340
330
320
(b)

LSD 0.05p= 1.085


a

Number of grains spike

-1

44
43
42

b
bc

41
c

40
39
38
37

(c)

LSD 0.05p= 0.2153


43

1000 grain weight (g)

42
b

41

40
39

38
37
36

.W
ist

er
at
l
til

t)
g(
ir n
e
t+

j)
g(
tin
n
i
Jo
t+

b)
g(
it n
o
bo
j+

t+

ng
di
ea
h
b+
j+

g
in
ad
he

Stages of MLE foliar spray


Fig. 4.5.3. Effect of exogenous application of MLE on number of fertile tillers

(a), number of grains per spike (b) and 1000 grain weight (c) of
wheat cv. Sehar-2006 under late sown conditions.

98

(a)

LSD 0.05p= 0.019

-1

Biological yield (t ha )

15.0

13.0
11.0
9.0
7.0
5.0
3.0
1.0

(b)

LSD 0.05p= 0.019

-1

Grain yield (t ha )

6.0
4.5
c

3.0
1.5
0.0

(c)

LSD 0.05p= 0.115

25

bc

Harvest index (%)

20
15
10
5

j+
b+
h
t+

he
ad
in
g

ea
di
ng

(b
)
oo
tin
g
t+
j+
b

Jo
in
tin
g(
j)
t+

(t)
til
le
rin
g

D
ist
.W

at
er

Stages of MLE foliar spray


Fig. 4.5.4. Effect of exogenous application of MLE on biological yield (a),

grain yield (b) and harvest index of wheat cv. Sehar-2006 under late
sown conditions.

99

4.6. Experiment VI:


Response of wheat to exogenous application of MLE under saline stress
conditions
The growth, yield and antioxidant status of wheat was studied as affected by exogenous
application of MLE and other growth enhancers under saline conditions. The data collected
during course of research are mentioned and discussed below:
The increased salinity significantly affected the shoot and root growth of wheat seedlings
(Table 4. 6.1 and 4.6.2). The reduction in shoot length while enhancement in root length was
found with increased salinity (Fig. 4.6.1 a, d). BAP and MLE foliar spray showed maximum
shoot length under low saline conditions while the longest roots were observed with similar
treatments under highly saline conditions. All the treatments produced shoot fresh and dry
weight better than control under each salinity levels (Fig. 4.6.1 b, c). The exogenous
application of BAP and MLE improved shoot fresh and dry weight as compared to control
under moderate or high salinity levels. Imposition of salt stress also reduced biomass
accumulation in roots (Fig. 4.6.1 e, f). The exogenous application of different growth
enhancers minimized the effect of salinity under moderate and high salinity levels. As a
result more root fresh and dry weights as compared to control were observed under 8 and 12
dS m-1 by BAP and MLE foliar spray, respectively. The analysis of variance for the leaf area
indicates a significant difference among plants treated with exogenous application of
different growth enhancers under salinity (Table 4.6.3). The largest leaf area was produced
by MLE application under less saline conditions (Fig. 4.6.2 a). While BAP performed better
than other growth enhancers under moderate or high salinity as exhibited by more leaf area.
The chlorophyll a and b contents were decreased by salinity. Under nonsaline conditions
highest contents were obtained from MLE treated plants (Fig. 4.6.2 b, c) whereas in saline
condition either moderate or high BAP showed maximum enhancement in the chlorophyll
contents.
The MLE and BAP were statistically at par with respect to total soluble proteins (Fig. 4.6.3
a). The protein contents were increased with increasing salinity stress and more protein being
observed at highest salinity level. At 12 dS m-1 largest total soluble proteins observed in BAP
foliar spray and minimum total soluble proteins produced in control under low saline
conditions. Both the qualitative and quantitative difference in antioxidant contents were
100

observed in salinized medium in response to application of growth enhancers (Fig. 4.6.3 a, b,


c, d, e, f). Increasing salinity increased the contents of enzymatic antioxidants up to 8 dS m-1.
MLE treatment showed higher content of enzymatic antioxidants i.e. catalase, peroxidase and
superoxide dismutase (SOD) as compared to control under low and moderately saline
conditions. The higher salinity levels caused a reduction in enzymatic antioxidant contents
and minimum effects were observed in MLE foliar spray. The minimum recorded value of
SOD, POD and catalase were under less saline conditions in control treatment. In non
enzymatic antioxidant salt stress showed a gradual rise in ascorbic acid contents (Fig.
4.6.3c). The significantly maximum content of ascorbic acid was obtained by MLE treated
plants under highly saline conditions. Lowest ascorbic acid was found in control under
normal conditions. The higher salinity showed phenolics lesser than moderate salinity but
better than less saline conditions. The peak value of total phenolic content was observed
under MLE foliar application at moderate salinity. The minimum phenolics value was
observed at 4 dS m-1 in case of control. Accumulation of Na+ and Cl- in the leaves was
significantly increased under saline conditions (Fig 4.6.4 a, c). Exogenous application of
MLE showed minimum Na+ and Cl- with maximum K+ in leaves under highly saline
conditions. The maximum Cl- contents were also found in control but under highly saline
conditions (Fig. 4.6.4 c.). As far as yield attributes are concerned the increasing salinity
show a decreasing trend in number of grains and 100 grain weight (Fig. 4.6.5 a, b). The BAP
application produced maximum number of grains whereas highest grain weight was obtained
under MLE treated plants in less saline conditions. The minimum number of grains and 100
grain weight was observed in control at 12 dS m-1.
Discussion
It is widely believed that salt stress greatly reduces wheat growth at all growth stages,
particularly during germination and seedling stage (Munns et al., 2006). Suppression of
growth in different plant organs occurs differently under saline conditions (Shoresh et al.,
2011). Salinity caused similar reduction in biomass of both root and shoot (fresh and dry
weight) but proportionally shoots length was decreased more as compared to roots length in
present study. However, exogenous application of essential elements enriched MLE through
foliar spray was found promising to accelerate the growth of different plant organs both
under saline and non saline conditions. Previously, reduction in growth of all the vegetative,

101

reproductive and biochemical parameters of tomato was found proportional with increasing
salinity of irrigation but foliar application of essential minerals like K, Fe and B minimized
the deleterious effects of salinity up to various extents. In the same study spray of individual
mineral was less effective as compared with their mixture but still better than non spray
control (Azeem and Ahmad, 2011).
The yield of a crop is mostly affected by its photosynthetic ability (Akram et al., 2002). The
smallest leaf area observed in present investigation under highest salinity level may be due to
reduction in water uptake and the nutritional imbalance causing toxicities or deficiencies of
ions under salinity leading to leaf injuries (Munns et al., 2006). The salinity not only reduced
the leaf area but also decreased chlorophyll contents. Nevertheless, the foliar spray of growth
enhancers partially alleviates this reduction and minimum reduction in chlorophyll contents
was observed with foliar spray of MLE during moderate salinity. Shoresh et al. (2011)
observed that salinity caused reduction in chlorophyll content in combination with the
reduced leaf area and growth. He further found that negative effects of salinity can be
ameliorated partially with improvement of plant growth by supplemental Ca2+ application. It
has been reported that moringa leaves have four times more calcium than milk, three times
the potassium of banana and seven times the ascorbic acid of oranges (Foidle et al., 2001), so
the application of MLE improve chlorophyll contents under salinity. Hanaa et al. (2008) also
earlier reported that total chlorophyll, chlorophyll a and chlorophyll b contents were
significantly improved in wheat plants sprayed with bioregulator (bezyladenine and ascorbic
acid), but this increase was less pronounced, when compared with that induced in wheat
stressed plant treated with algal antioxidant extracts. While foliar spray of ascorbic acid also
minimized the reduction of chlorophyll a by salinity in wheat (Athar et al., 2008). Foliar
application of potassium containing materials can minimize the antagonistic effects of soil
salinity (Ahmad and Jabeen, 2005). Moringa leaf extracts foliar application prevents
premature leaf senescence and resulting in more leaf area with higher photosynthetic
pigments (Rehman and Basra, 2010).
The incomplete reduction of O2 to form water under abiotic stresses such as salinity leads to
generation of reactive oxygen species (ROS) such as singlet oxygen, superoxide (O2),
hydrogen peroxide (H2O2) and hydroxyl radical (OH) (Mittler, 2002; Dat et al., 2000). As
they are in ionic forms or being radicals so highly reactive and a threat to the cell membrane

102

and other important cellular organelle DNA, proteins and lipids etc. In plant defense
mechanism both enzymatic (Superoxide dismutase (SOD), Catalase (CAT), Peroxidase
(POD) etc.) and non enzymatic (ascorbates, phenols etc.) antioxidants as scavenger of ROS
have gained primary consideration (Asada and Takahashi, 1987; Noctor and Foyer, 1998;
Sgherri et al., 2004). We found enhancement in antioxidants contents under exogenous
application of growth enhancers was more at 8 dS m-1 as compared to 12 dS m-1 MLE exhibit
highest antioxidant status. The continuous improvements in ascorbic acid contents with
increasing salinity were obtained by MLE application. Ascorbates are powerful primary
antioxidant and direct scavenger of ROS (Buettner and Jurkiewicz, 1996). Ascorbic acid
induced ionic changes might have triggered the antioxidant system. Thus, enhanced salt
tolerance in wheat plants was due to ascorbic acid producing a better antioxidant system for
the effective removal of ROS in plants, and help in maintenance of ion homeostasis (Mittler,
2002). Overall, exogenous application of ascorbate increased endogenous level of ascorbic
acid which exerted a protective effect on growth and also improved photosynthetic capacity
of wheat against salt induced oxidative stress (Athar et al., 2008). The increased contents of
antioxidants in response to enhanced level of ROS generation under salinity have been
studied in wheat, cotton, maize and rice (Sairam et al., 2001; Vaidyanathan et al., 2003).
According to Zhang and Ervin (2008) stress tolerance in creeping bent grass was improved
with increased SOD level by the application of cytokinin containing sea weed extract. Since
moringa is a rich source of zeatin (Foidle et al., 2001), so the effectiveness of MLE in
mitigating salinity by better chlorophyll, antioxidants and plant growth might be due to
cytokinin mediated stay green effect. Similarly, H2O2 exogenous application also plays an
important role in improving plants tolerance against oxidative stress by activating plant
antioxidants system (Gechev et al., 2002).
Salt tolerance in sugarcane leaves improved with enhanced level of soluble phenolics
(Wahid and Ghazanfar, 2006). It may be justified that under moderate salinity ROS were
cellular indicators playing roles of secondary messengers to activate plant defense system
against salt injuries (Knight and Knight, 2001) resulting in increased antioxidants level by
these treatment. But under highly saline conditions injuries caused by ROS exceeded their
intracellular stress monitoring role and resulted in enzyme inhibition, oxidation and
peroxidation of proteins and lipids respectively leading to programmed cell death (Mittler,

103

2002). So the lesser quantities of antioxidants at 12 dS m-1 as compared to 8 dS m-1 were


found except ascorbates which continue to increase up to 12 dS m-1.
Salinity also produces ionic stress leading to ionic imbalances and impairment of root
membrane selectivity. In our research the application of MLE, BAP and H2O2 decreased the
concentration of Na+ and Cl- along with increased concentration of K+ in leaves against
control. The maximum exclusion of Na+ and Cl- with highest K+ accumulation was observed
in MLE foliar spray up to moderate salt level. Similarly, increased K+ accumulation in leaves
under salinity was obtained by ascorbic acid application in wheat (Athar et al., 2008).
Makkar et al. (2007) reported that MLE contains significant quantities of calcium, potassium,
and cytokinin in the form of zeatin, antioxidants proteins, ascorbates and phenols. Such
compositions of MLE not only enhanced seedling vigor and provide a good start to plant
even under saline conditions but also improved the grain yield.

104

Table 4.6.1 Mean sum of squares of the data for shoot length, shoot fresh weight and
shoot dry weight of wheat by exogenous application of growth enhancers under saline
conditions.
SOV

Df

Shoot length

Shoot fresh weight

Shoot dry weight

Salinity

100.107**

9.003**

1.581**

Treatment

38.547**

0.433**

0.198**

2.698**

0.047**

0.016**

24

1.036

0.011

0.000

Salinity
Treatment
Error

Table 4.6.2 Mean sum of squares of data for root length, root fresh weight and root dry
weight of wheat by exogenous application of growth enhancers under saline conditions.
SOV

Df

Root length

Root fresh weight

Root dry weight

Salinity

98.045**

3.096**

0.184**

Treatment

30.592**

0.655**

0.028**

1.110

0.026**

0.002**

24

1.738

0.004

0.000

Salinity
Treatment
Error
** p < 0.01

ns = non significant

105

Table 4.6.3 Mean sum of squares of the data for leaf area, leaf chlorophyll a and chlorophyll
b of wheat by exogenous application of growth enhancers under saline conditions.
SOV

df

Leaf area

Chlorophyll a

Chlorophyll b

Salinity

1333.117**

0.259**

0.010**

Treatment

745.538**

0.290**

0.003**

35.512**

0.003**

0.000**

24

1.437

0.000

0.000

Salinity
Treatment
Error

Table 4.6.4 Mean sum of squares of the data for total soluble protein, enzymatic antioxidants
SOD and POD of wheat by exogenous application of growth enhancers under saline conditions.
SOV

Df

Total soluble

Superoxide

Peroxidase

protein

dismutase (SOD)

(POD)

Salinity

0.012**

4076.635**

51.660**

Treatment

0.040**

16694.870**

272.886**

0.000*

771.991**

8.307**

24

0.000

15.810

0.340

Salinity
Treatment
Error

** p < 0.01

ns = non significant

106

Table 4.6.5 Mean sum of squares of the data for enzymatic antioxidants (Catalase),
non- enzymatic antioxidants i.e. total phenolic contents and ascorbic acid of wheat by
exogenous application of growth enhancers under saline conditions.
SOV

Df

Catalase

Total phenolic content

(CAT)

(TPC)

Ascorbic acid

Salinity

24.339**

0.173**

0.023**

Treatment

38.528**

3.229**

0.126**

2.629**

0.032**

0.011**

24

0.040

0.008

0.000

Salinity
Treatment
Error

Table 4.6.6 Mean sum of square of the data for Na+, K+ and CI- contents of wheat by
exogenous application of growth enhancers under saline conditions.
Df

Na+

K+

Cl-

Salinity

0.036**

0.151**

5.510**

Treatment

0.161**

0.735**

8.680**

0.247**

0.106**

2.534**

24

0.001

0.002

0.034

SOV

Salinity
Treatment
Error
** p < 0.01

ns = non significant

107

Table 4.6.7 Mean sum of squares of the data for number of grains per spike and 100
grain weight of wheat by exogenous application of growth enhancers under saline
conditions.
SOV

Df

Number of
grains per spike

100 grain weight

--

Salinity

1043.084**

1.451**

--

Treatment

353.688**

1.154**

--

Salinity x
Treatment
Error

17.847**

0.032**

24

1.084

0.008

** p < 0.01

ns = non significant

108

---

(a)

LSD 0.05p=1.715
Control
BAP foliar spray

60.0
50.0

50.0
ab a
bc
cde

bc
cd cde

ef

de

30.0

Root length (cm)

Shoot length (cm

60.0

40.0

20.0

0.0
8

(b)

12

40.0
30.0
20.0

(e)

LSD 0.05p=0.1060

4.5

4.5
a a

4.0
Root fresh weight (g)

4.0

3.0
2.5

c c

d
e e

2.0

ef

1.5
1.0

3.5
2.5

12
(c)

f f

1.0
0.0

LSD 0.05p=0.05329

d d e

1.5

0.0
8

2.0

0.5
4

a b

3.0

0.5

12
(f)

LSD 0.05p=0.05329

1.50

1.50

a a

Root dry weight (g)

Shoot fresh weight (g)

ab a cd
cde

12

LSD 0.05p=0.1767

Shoot dry weight (g)

ab a cd
cde

0.0
4

1.25

bc
de bc cde

10.0

10.0

3.5

(d)

LSD 0.05p=2.222

MLE foliar spray


H2O2 foliar spray

d d

1.00
f

0.75

e
f f

0.50
h

1.25
1.00
b

bc bc c
e

0.25

0.00

0.00
4

8
12
Salinity (dS m-1)

b
d

0.50

0.25

a a

0.75

d d d

8
Salinity (dS m-1)

Fig 4.6.1. Effect of exogenous application of different plant growth enhancer on

shoot length (a), shoot fresh weight (b), shoot dry weight (c), root
length (d), root fresh weight (e) and root dry weight (f) of wheat cv.
Sehar-2006 under saline conditions

109

12

(a)

LSD 0.05p=2.020
Control
BAP foliar spray

a b

80.0

-2

Leaf area (cm )

70.0

MLE foliar spray


H2O2 foliar spray

c
d

60.0

g h

50.0
40.0

30.0
20.0
10.0
0.0
4

12
(b)

LSD 0.05p=0.05329
1.00

a a

-1

Chlorophyll a (mg g )

bc c

0.75
d
0.50

d d
e

e
f
g

0.25

0.00
4

12
(c)

LSD 0.05p=0.05329

-1

Chlorophyll b (mg g )

1.00
0.75
0.50
0.25

ab a a ab

bc

ab

ab

ab

abc
abc bc
c

0.00
4

8
Salinity (dS m-1)

12

Fig 4.6.2. Effect of exogenous application of different plant growth enhancers

on leaf area (a), chlorophyll a (b) and chlorophyll b (c) of wheat cv.
Sehar-2006 under saline conditions

110

(a)

LSD 0.05p= 0.05329

2.0
1.5
1.0

a
cdabc d

0.5

de ab

abc
e

ab
abc bc

0.0
4

(b)

hi

1.0
0.0
8

12
(e)

-1

20
15

25

de
e

f f

f
g

10
5
0

12

4
(c)

LSD 0.05p= 0.05329

-1

a
b

0.75
d
0.50

ef de
h

d d
fg

gh

0.25

4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

8
Salinity (dS m-1)

12

12
(f)

a bc

cd d

b cd
e

0.00

LSD 0.05p= 0.1507


-1

1.00
Ascorbic acid (m.mol g )

h
2.0

-1

POD (IU min mg protien)

f f
cd

LSD 0.05p= 0.9826

Total phenolic contents (mg g )

-1
-1

a b

30
c

f ef

c d
3.0

a b

180
160
140
120
100
80
60
40
20
0

4.0

12

LSD 0.05p= ns

SOD (IU min mg protien)

Catalase (IU min-1 mg-1 protien)

MLE foliar spray


H2O2 foliar spray

-1

Total soluble protein (mg g )

Control
BAP foliar spray

(d)

LSD 0.05p= 0.3370

fg

12
-1

Salinity (dS m )

Fig 4.6.3. Effect of exogenous application of different plant growth enhancers

on total soluble protein (a), SOD (b), ascorbic acid (c), catalase (d),
POD (e) and total phenolics (f) of wheat cv. Sehar-2006 under saline
conditions

111

fg

(a)

LSD 0.05p= 0.05329


Control
BAP foliar spray

MLE foliar spray


H2O2 foliar spray

-1

1.5
1.0

Na (mg g dry weight)

2.0

0.5

d de

ef

0.0
4

12
(b)

LSD 0.05p= 0.05329

a
1.5

b b

b
c

1.0

b d

cb

e
g

-1

K (mg g dry weight)

2.0

0.5

0.0
4

12
(c)

LSD 0.05p= 0.3107

10.0
8.0

g e

6.0
4.0

-1

Cl (mg g dry weight)

12.0

2.0
0.0
4

12
-1

Salanity (dS m )

Fig 4.6.4. Effect of exogenous application of different plant growth enhancer on

leaf Na+ (a), K+ (b) and Cl- (c) of wheat cv. Sehar-2006 under saline
conditions

112

(a)

LSD 0.05p= 1.755


Control
BAP foliar spray

60
-1

Number of grains spike

40

50

MLE foliar spray


H2O2 foliar spray

c
d

f
g

30

g
i

20
10
0
4

12
(b)

LSD 0.05p= 0.1507

100 grain weight (g)

3.5
3.0

a a
b

de

b cd

bc ef
f

2.5

gh

2.0

1.5
1.0
0.5
0.0
4

8
Salinity (dS m-1)

12

Fig 4.6.5. Effect of exogenous application of different plant growth enhancers

on number of grains (a) and 100 grain weight (b) of wheat cv. Sehar2006 under saline conditions

113

4.7. Experiment VII: Response of wheat to exogenous application of MLE


under drought stress conditions
Increasing drought stress levels significantly affected plant growth and yield (Table 4.7.1 and
4.7.4). But improvement in antioxidants status was obtained by exogenous applications
(Table 4.7.2, 4.7.3). The detailed results are mentioned and discussed below:
Comparison of means indicate that leaf area was gradually reduced with increasing drought
stress (Fig. 4.7.1a). The minimum leaf area was obtained under extreme drought conditions.
The interaction of drought and foliar application on leaf area was found to be significant
(Table 4.7.1). The leaf area produced by the foliar application of MLE and BAP was better
than control under well watered, moderate and severe drought stress. The minimum leaf area
was recorded in control under severe drought stress.
The increasing water stress decreased leaf chlorophyll a contents (Fig. 4.7.1 b). The
maximum quantity of chlorophyll a was observed under well watered conditions by
exogenous application of MLE and BAP. While in moderate and severe drought, foliar
application of K and MLE exhibit higher and statistically similar leaf chlorophyll a. The
similar trend were followed by all the treatments in case of chlorophyll b under each level of
water stress (Fig. 4.7.1 a). The minimum quantity of chlorophyll a and chlorophyll b were
produced by control under extreme water stress.
A direct relationship was evident between drought stress level and leaf total soluble protein
(Fig. 4.7.2 a). The largest leaf protein content was produced at moderate followed by severe
with minimum value under well watered conditions. The BAP foliar spray showed
significantly highest value of leaf protein in well watered and under both drought stress
levels. Nevertheless, the exogenous application of growth enhancers performed better than
control in case of leaf total soluble protein.
The rise in drought stress amplified the antioxidants status of wheat plant (Fig. 4.7.2 b, c, d).
The enzymatic and nonenzymatic antioxidants contents produced by exogenous application
of all growth enhancers were better than control under each water level. The contents of
enzymatic antioxidants were increased up to moderate drought stress. The application of
BAP produced maximum superoxide dismutase (SOD) under severe drought. In case of
POD, water stress increased POD contents as compared to non stress conditions. Largest

114

increase was found under medium drought stress by MLE application. Similar trend as that
of POD was followed by catalase.
Decreasing water level increased ascorbic acid contents in all the treatments (Fig. 4.7.2 e).
The highest value of ascorbic acid obtained under moderate drought by exogenous
application of MLE30. However, ascorbic acid remains unaffected by severity of drought and
produced in similar quantity under moderate and severe drought by foliar application of
MLE30.
The TPC contents were found in comparatively lesser quantities as compared to ascorbic
acid (Fig. 4.7.2 e). The maximum TPC produced during severe water stress under MLE30.
The application of K+ and BAP showed similar TPC contents under well watered and severe
drought. The minimum TPC were obtained in case of control under well watered conditions
MLE application accumulated largest leaf K+ ions under moderate drought stress (Fig. 4.7.3
a). Under severe water stress foliar application of MLE produced K+ ion in similar quantity
as that of its respective value under well watered conditions. The least K+ ion was observed
in control under severe drought stress.
Highest grain yield plant-1 observed in the absence of water stress by MLE foliar spray (Fig.
4.7.3b). However, deficiency of water decreased grain yield but all the treatments
ameliorated the negative effects of water stress significantly better than control. MLE
application produced more grain yield under moderate and severe water stress as compared
to BAP, K+ and control.
Discussion
There is a faster expansion in arid zones on our planet as an impact of climate change. The
dual challenges of expanding arid land and faster worlds population growth exerts direct
impact on water reservoirs and water availability. This aridity and water stress have direct
impact on crop growth and yield reduction. We also reported that water stress caused
reduction in growth and grain yield of wheat. Reduction in plant growth under restricted
water availability might have been due to reduction in photosynthesizing tissue and
photosynthetic capacity (Athar and Ashraf, 2005; Chaves et al., 2011). Chlorophyll and
protein contents affect the photosynthetic capacity of plants and considered as important
adaptation traits under drought stress conditions (Chernyadev and Monakhova, 2003). The
level of chlorophyll must be essentially maintained in water deficit situation for supporting

115

photosynthetic capacity of plants (Sairam et al., 1997). Nevertheless, in the present study,
reduced yield due to water stress was not only correlated with leaf area but also with
chlorophyll a and chlorophyll b (Fig. 4.7.1). Exogenous application caused increase in yield
under normal or water stress conditions, except that of K+ as a foliar spray. However, foliar
application of MLE caused maximum increase in grain yield of wheat under control or water
stress conditions. These results are similar with those of Zhang et al. (2004) who found that
chlorophyll contents were significantly improved by exogenous application of growth
regulators under water deficit conditions. The foliar application of algal extract, MLE and
BAP may also stimulate earlier cytokinin formation thus preventing premature leaf
senescence resulting in more leaf area with higher photosynthetic pigments (Hanaa et al.,
2008; Rehman and Basra, 2010; Ali et al., 2011). The phytoregulators exhibiting cytokinin
activity showed protective effects in water deficit and prevent reduction in chlorophyll and
protein contents. In view of Schachtman and Goodger (2008) plant growth regulators such as
cytokinins, activated under water stress conditions initiate defensive responses in plants to
protect plants important processes from water stress injuries. In the presence of cytokinin
like activity compounds e.g. Kartolin less reduction were observed in protein contents
(Chernyadev and Monakhova, 2003).The positive effects of BAP on protein pool was also
pronounced under drought stress. Besides adaptation role, hormones also regulate yield
potential, by controlling floret survival and grain filling capacity in cereals (Young et al.,
1997). Less reduction in growth and yield observed in present study by foliar spray of MLE
and BAP in drought stressed wheat plants could be attributed to hormonal influence
especially rich zeatin contents of moringa.
Moringa leaves have been reported to be a rich source of -carotene, protein, vitamin C,
calcium and potassium and act as a good source of natural antioxidants such as ascorbic
acid, flavonoid, phenolics and carotenoids (Dillard and German, 2000; Siddhuraju and
Becker, 2003). So the exogenous applications of MLE improve the antioxidant status and
yield of wheat under drought stress.

116

Table 4.7.1 Mean sum of squares of data for leaf area, leaf chlorophyll a and
chlorophyll b contents of wheat by exogenous application of growth enhancers under
drought conditions.
SOV

df

Chlorophyll a

Chlorophyll b

Leaf area

Drought

0.323**

0.293**

21294.976**

Treatments

0.726**

0.028**

23144.557**

Drought x
Treatments
Error

0.064**

0.004**

848.006**

24

0.001

0.001

51.686

Table 4.7.2 Mean sum of squares of data for total soluble proteins, enzymatic
antioxidants i.e. super oxide dismutase (SOD) and peroxidase (POD) of wheat by
exogenous application of growth enhancers under drought conditions.
SOV

df

Total soluble
proteins

Superoxide
dismutase (SOD)

Peroxidase (POD)

Drought

0.052**

158.854**

5.973**

Treatments

0.511**

523.489**

7.031**

Drought x
Treatments
Error

0.025**

17.088**

0.681**

24

0.004

1.338

0.013

** P < 0.01

117

Table 4.7.3 Mean sum of squares of data for enzymatic antioxidant catalase (CAT),
non-enzymatic antioxidant ascorbic acid and total phenolics (TPC) of wheat by
exogenous application of growth enhancers under drought conditions.
SOV

df

Catalase (CAT)

Ascorbic acid

Total phenolics (TPC)

Drought

1357.425**

364.912**

2.689**

Treatments

642.191**

486.082**

6.214**

Drought x
Treatments
Error

64.105**

15.383**

0.208**

24

1.550

0.819

0.013

Table 4.7.4 Mean sum of squares of data for leaf K+ contents and grain yield of wheat
by exogenous application of growth enhancers under drought conditions.
Leaf K+ contents

Grain yield per

SOV

df

Drought

0.111**

1.924**

Treatments

0.287**

0.265**

Drought x
Treatments
Error

0.008**

0.007*

24

0.001

0.004

plant

** P < 0.01

118

(a)

LSD 0.05p = 12.12


Control
BAP foliar spray

a a

500.0
Leaf area (cm2)

MLE30 foliar spray


K (2%) foliar spray

400.0

b b
d d e

d
f
g

300.0
200.0
100.0
0.0
100

75

50
(b)

LSD 0.05p = 0.05329

Chlorophyll a (mg g-1 fw)

2.5
2.0

a ab
c

bc

cd

de

ed
h

1.5
i
j
1.0
0.5
0.0
100

75

50
(c)

Chlorophyll b (mg g-1 fw)

LSD 0.05p = 0.05329


1.5

1.0
a a
bc

cd
fg

0.5

ef de

fg
h

0.0
100

75

50

Field capacity (%)

Fig.4.7.1. Effect of exogenous application of different plant growth enhancers

on leaf area (a), chlorophyll a (b) and chlorophyll b (c) of wheat cv.
Sehar-2006 under drought conditions.

119

2.5
2.0

b ab

1.5

1.0
0.5

40.0
30.0

c
e
h

d
g

20.0
10.0
0.0

75

50

100
(b)

LSD 0.05p = 0.1066

75

50
(e)

LSD 0.05p = 0.1921

3.0
2.5
2.0

1.5

b ab

1.0
0.5
0.0

POD (IU mg-1 min-1 protien)

10.0
8.0
a

e
f

fg g fg
4.0

6.0
h

2.0
0.0

100

75

50
(c)

LSD 0.05p = 1.525


50.0

b c
d

30.0
20.0

40.0

100

fg

10.0
0.0

75

50
(f)

LSD 0.05p = 0.1921


Total phenolic content (mg
GAE g-1)

SOD (IU mg-1 min-1 protien)

a
60.0
50.0

0.0
100

Ascorbic acid (m.mol g-1)

70.0

-1

3.0

min protien)

MLE30 foliar spray


K (2%) foliar spray

-1

Total soluble protein (mg g-1 fw)

Control
BAP foliar spray

(d)

LSD 0.05p = 2.098

Catalase (IU mg

(a)

LSD 0.05p = 0.1066

5.0
a
4.0
e

3.0
2.0

c d
f f

b bc
e
g

1.0
0.0

100

75

50

100

Field capacity (%)

75
50
Field capacity (%)

Fig.4.7.2. Effect of exogenous application of different plant growth enhancer

on total soluble protein (a), super oxide dismutase (b), ascorbic acid
(c), catalase (d), peroxidase (e) and total phenolic contents (f) of
wheat cv. Sehar-2006 crop under drought conditions.

120

(a)

LSD 0.05p = 0.05329

Leaf K+ (mg g-1 dry weight)

Control
BAP foliar spray

MLE30 foliar spray


K (2%) foliar spray

2.0
a
1.5
1.0

c d

b b

d cd

0.5
0.0
100

75

50
(b)

LSD 0.05p = 0.1066

Grain yield plant-1

2.5
2.0

a ab
b
c

cd

d d
e

1.5

f f

1.0
0.5
0.0
100

75

50

Field capacity (%)

Fig.4.7.3. Effect of exogenous application of different plant growth enhancer


+
-1
on leaf K contents (a) and grain yield plant (b) of wheat cv.
Sehar-2006 under drought conditions.

121

General Discussion
This dissertation examines the potential of moringa leaf extract (MLE) as a natural plant
growth enhancer in various crops under normal and abiotic stresses. Moringa belongs to
family Moringaceae and is a natural as well as cultivated variety of the genus Moringa
(Mahmood et al., 2010). It is the richest plant source of vitamins A, B, C, D, E and K (Anwar
and Bhanger, 2003; Babu, 2000; Caceres et al., 1992; Dayrit et al., 1990). The vital minerals
present in moringa leaves include calcium, potassium, copper, iron, magnesium, zinc and
manganese. From the analysis of moringa leaves it become clear that they have appreciable
quantities of mineral nitrogen, phosphorus, potassium, calcium, magnesium, zinc, copper,
iron, manganese (Table, 3.1). Moreover presence of total soluble protein, enzymatic
antioxidants (SOD, POD and CAT) and non enzymatic antioxidants (total phenolics and
ascorbates) was observed during analysis of moringa leaf extract (Table, 3.1). Foidle et al.
(2001) also reported that MLE have plant growth enhancing capabilities as it is rich in zeatin,
ascorbates, carotenoids, phenols, potassium and calcium. Moringa truly appears a gift of
nature and a "Miracle" plant having countless benefits for humanity so it can be used to fight
against hunger, poverty and malnutrition.
Leaf extracts of M. oleifera (MLE) have been reported to accelerate growth of tomato,
peanut, corn and wheat at early vegetative growth stage, improve resistance to pests and
diseases and enhanced more and larger fruits and generally increase yield by 20 to 35%
(Fuglie, 2000). The germination and growth promotion of wheat seeds was found to be
concentration depended and MLE diluted up to 30 times proved effective as compared to
other dilutions. The reduced germination of wheat seeds by application of concentrated (10 times
diluted) MLE was also reported by Phiri (2010). The growth enhancing characteristics of MLE was
also noted to be affected by its method of exogenous application (priming, soil or foliar). In some

earlier studies it has been observed that seed priming with plant growth regulators, inorganic
salts, compatible solutes or sugar beet extract caused improvement in seed germination by
providing physiological and biochemical adaptations (Pill and Savage, 2008; Afzal et al.,
2006a). The seed priming with MLE30 enhances seed germination and seedling vigour along
with improvement in antioxidant status of wheat. Likewise, while working with three range
grasses (Cenchrus ciliaris, Panicum antidotale and Echinochloa crusgalli) Nouman et al.
(2011) reported that seed priming with MLE30 significantly increased germination of all the
122

range grasses. It may be attributed that MLE is a rich source of zeatin, ascorbic acid, Ca2+,
and K+ (Fuglie 1999; Foidle et al. 2001), which are involved in improving several plant
growth and development processes. Al-Hakimi and Hamada (2001) observed that seed
priming with ascorbic acid increased leaf soluble proteins. According to Price (2000)
moringa leaf extract contain ascorbic acid in appreciable quantities. Thus, increased leaf
protein due to MLE30 seed priming was one of the reasons that contributed in improved
growth of wheat. The foliar applied MLE30 as compared to soil application caused more
enhancements in antioxidants, growth and yield of tomato and pea crop.
Besides crop production under normal conditions, abiotic stresses cause significant crop
losses with the 50% reduction in average yield of major crops (Bray et al., 2000). Among
abiotic stresses drought, salinity and extreme temperature along with chemical toxicity and
oxidative stress becoming major threats to agriculture and natural environment sustainability
(Wang et al., 2003). As plants produce significant amount of antioxidants to prevent the
oxidative stress, they represent a potential source of compounds with antioxidant activity
such as ascorbic acid, total phenols, and vitamins in addition to mineral elements K+, Ca2+
and PGRs. Alternatively, toxicological effects of synthetic antioxidants and consumer
preference for natural products have resulted in increased interest in the application of natural
antioxidants (Castenmiller et al., 2002; Kaur and Kapoor, 2001; Koleva et al., 2002; Pizzale
et al., 2002). Siddhuraju and Becker (2003) observed antioxidant properties in the solvent
extract of moringa leaves and reported that leaves are potential source of natural antioxidants.
According to Arabshahi et al. (2007) the extracts from drumstick and carrot had a higher
antioxidant activity (83% and 80%) than -tocopherol (72%). The presence of antioxidants in
MLE was also observed during current studies (Table 3.1).
Under late planting of wheat, maintenance of photosynthetic activity due to increased
temperatures during maturation (Paulsen, 1994) and efficient utilization of these
photosynthates indicated by high harvest index, (Gifford and Thorne, 1984; Blum et al.,
1994) are two important determinants of grain yield. The maximum recorded harvest index
by MLE sprayed from start up to end of wheat growth (tillering + jointing + booting +
heading) supported that photosynthetic activity was maintained up to maturity which was
result of longer seasonal leaf area duration and more stay green period. This delayed onset of
leaf senescence is reported to provoke about 11% more carbon fixation in Lolium temulentum

123

(Thomas and Howarth, 2000). An extension in active photosynthetic period may enhance
total photosynthates availability in annual crops life cycle and higher mass per grain can be
achieved if assimilated carbon supply be maintained to grain during grain filling period
(Spano et al., 2003). MLE extended the seasonal leaf area duration (SLAD) by 9.22 d and
6.45 d over control when applied at all growth stages and single spray at heading,
respectively.
The increased contents of antioxidants in response to enhanced level of ROS generation
under salinity have been studied in wheat, cotton, maize and rice (Sairam et al., 2001;
Vaidyanathan et al., 2003). The foliar application of MLE exhibit highest antioxidant status
as compared to other growth enhancers up to moderate salinity (8 dS m-1) except ascorbic
acid which continued to be increased up to highest salinity level (12 dS m-1). Moreover, the
increase in ascorbate under MLE application in salinity stress was largest than all other
enzymatic and nonenzymatic antioxidants. As reported earlier MLE is a rich source of
ascorbate and exogenous application of ascorbate increased endogenous level of ascorbic
acid which exerted a protective effect on growth and also improved photosynthetic capacity
of wheat against salt induced oxidative stress (Athar et al., 2008). The phytoregulators
exhibiting cytokinin activity showed protective effects in water deficit and prevent reduction
in chlorophyll and protein contents. In view of Schachtman and Goodger (2008) plant growth
regulators such as cytokinins, activated under water stress conditions initiate defensive
responses in plants to protect plants important processes from water stress injuries. Moringa
leaves have been reported to be a rich source of -carotene, protein, vitamin C, calcium and
potassium and act as a good source of natural antioxidants such as ascorbic acid, flavonoid,
phenolics, carotenoids and PGR like zeatin (Dillard and German, 2000; Siddhuraju and
Becker, 2003). So the exogenous applications of MLE improve the antioxidant status and
yield of wheat under drought stress.
Finally it is concluded that MLE was effective in improving growth and yield of wheat,
tomato and pea. Thirty times diluted MLE was found to be optimum dose in laboratory
studies and confirmed in pot and field studies. The exogenous application of MLE as priming
agent or foliar spray improve the antioxidant status chlorophyll contents, leaf area, grain
weight and finally yield of wheat crop under normal and abiotic stress (late sowing, drought
and salinity) conditions.

124

Summary
Crop productivity is affected by many abiotic stresses i.e. temperature extremes, drought and
salinity and each of these limit plant growth and development. Plants have internal system of
regulation to modify these responses. Plants also respond to the exogenous application of
antioxidants, PGRs and certain nutrients for abiotic and biotic stress tolerance that results in
higher economic return. This dissertation investigated the role of exogenous application of
MLE in vivo and in vitro as plant growth enhancer and amelioration of devastating affects of
high temperature stress in late sown wheat and under drought and salinity stresses. The
physiological and biochemical basis for improved performance of peas and tomato by MLE
application was also assessed. Wheat cultivar Sehar-2006, tomato cv.Sahil and pea
cv.Climax were used in the study. Seven independent experiments were performed to
examine the effect of exogenous application of MLE at the germination, seedling, vegetative
and adult growth stages. Experiments at the germination and seedling stages were conducted
in a laboratory, while those of at the adult growth stage were conducted in a wire-net house
and field conditions of the Department of Crop Physiology, University of Agriculture,
Faisalabad, Pakistan, during 2008-2009. In first experiment wheat seeds were allowed to
germinate supplemented with different dilutions of MLE. The second was comprised of
MLE evaluation as priming agent. The third and fourth experiment were involve the
assessment of MLE exogenous application through soil and foliar in tomato and pea. In the
fifth experiment potential of MLE was evaluated in growth and yield of wheat under late
sowing conditions. The sixth and seventh experiment was conducted in saline and drought
conditions respectively to assess the comparative effect of foliar applied growth enhancers
along with MLE. From the results of seed germination experiment it is evident that MLE30
(30 times diluted MLE) was the optimum dose. MLE30 priming excelled all other priming
agents used in study and showed 23% more grain weight which leads to highest (35%) grain
yield per plant with enhanced antioxidant status of plant. The foliar application was superior
to soil application; however both modes of exogenous application performed significantly
better than control in all growth, yield and biochemical parameters of tomato and pea crop.
The foliar application method of MLE30 dilution was most effective and has a potential to be
used as plant growth enhancer in tomato and pea crop. The foliar MLE spray delayed the
crop maturity, extend SLAD (9.22 d and 6.45 d over control) and grain filling period there by
125

leading to greater seed and biological yields in late sown wheat. The performance of MLE at
any growth stage under late sown wheat was better than control and the pronounced effects
of MLE were observed when it was sprayed at four critical crop stages (tillering + jointing +
booting + heading) with the maximum contribution being observed under MLE application
at heading. MLE foliar spray enhanced yield under normal conditions but more yield was
observed from MLE foliar spray in moderately saline conditions. MLE foliar application
produced more grain yield under 75% and 50% field capacity as compared to other
treatments in wheat.
In conclusion, MLE diluted up to 30 times was the most effective. MLE delayed senescence,
extended seasonal leaf area duration, leaf area, regulation of antioxidant system, improved
physiological parameters i.e. chlorophyll under abiotic stresses. Improvement in growth and
yield of wheat due to exogenous application of MLE under salinity and drought was the
result of improved antioxidant level and increased accumulation of K+ and reduced levels of
Na+ and Cl- in leaves.
These studies provide preliminary data and were first attempts to evaluate the potential of
MLE as growth enhancer under normal and stress conditions; however, more research is
needed to investigate the cause of enhancement. Moreover, further investigations are also
needed for quantification of cytokinin in MLE. The simple method of extraction and
exogenous application were used, so that it can be easily adopted by community, however,
more refinements like extraction methods and blending of MLE with other natural and
synthetic compounds, of theses techniques be required in future research.

126

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