Final Thesis

Morpho-Biochemical attributes of Moringa oleifera
Lam. of edaphically differential settings
A thesis submitted in partial fulfillment of the requirement for the degree

of Masters of Philosophy (M.phil) in the Subject of Botany
By
Nawal Idrees
Roll No: 06
Session: 2015-2018
(BOTANY DIVISION)
INSTITUTE OF PURE AND APPLIED BIOLOGY
BAHAUDDIN ZAKARIYA UNIVERSITY MULTAN
2018
1
CERTIFICATE
It is certified that Nawal Idrees Roll No 06 (2015-2018) has successfully completed her
research work contained in her thesis that had been carried out over 08 months in the partial
fulfillment of the requirement for the degree of Master of Philosophy (M.phil) in Botany at the
Institute of Pure and Applied Biology, Bahaudin Zakariya University, Multan Pakistan.
Supervisor:
(Prof. Dr. Seema Mahmood)
Director
(Prof .Dr.Muhammad Naeem)
Dated:
2
Chapter # 1 Introduction
Distribution
Moringa plant is aboriginal to northwestern India and also found in various countries of tropical
and sub-tropical regions of Africa, South East Asia and South America, Arabia. This plant have
several common names in different constituencies e.g. benzoil,cabbage tree, drumstick tree,
horseradish tree, kelor, marango, mlonge, mulangay, suhanjna and sajna(Fahey, 2005)
In Pakistan, Moringa is depicted by two species i.e. M. concanensis and M. oleifera. The former
species isn’t common and may be restricted to far off vicinity of Sindh. The latter, M. oleifera is
mature and cultivated in the Punjab plains, Sindh, Baluchistan, and North Western Frontier
Province , significantly temperate and tropical regions of the country (Anwar, 2003)
Taxonomy
Moringa oleifera belong to order Brassicales, family Moringaceae, genus Moringa. The
Moringaceae is a family with thirteen illustrious species with Moringa oleifera being the
foremost extensively used and utilized spp.(Mahmood, 2011).Moringa is a fast growing, small-
medium sized tree of 5-12m in height with an umbrella shaped canopy. The trunk is straight,
having corky whitish bark. It is an evergreen deciduous tree with tripinnate leaves. Flowers are
white to cream colored, nicely fragranced and zygomorphic. The mature fruit become 20-30cm
long and change its color from green to brown. Seeds are round or triangular with three papery
wings. It produces taproot. It’s a drought-tolerant, multi-purpose and one in all useful tree due to
its medicinal and nutritional properties in world and thus delineated as a ‘miracle
tree.(Arbonnier, 2002)
Traditional uses
Moringa oleifera is one of the ample plant sources of vitamins, minerals and anti-oxidants.
Studies have shown that It has more beta carotene, calcium, protein, potassium, vitamin C, iron
as compared to carrot, milk, pea, banana, orange, spinach in gram to gram comparison
respectively, therefore can be used as food supplement to reduce the effect of malnutrition(Goss,
2007). Moringa has been used by kings and queens in their diet for mental attentiveness and
3
healthy skin. All the parts e.g. leaves, pods, seeds, bark and flowers of moringa are used in more
than 80 countries because of its benefits. It is use to mitigate mineral and vitamin deficiencies,
maintain a healthy cardiovascular system, sustain normal blood-glucose levels, neutralize free
radicals by this means reduce malignancy, provide superb support to anti-inflammatory
mechanisms and immune system. It also helpful in improving eyesight and bone strength and
prove beneficial for lactating mothers, menopause, depression and osteoporosis. Along with
nutritional and therapeutic uses evidence are present showing the use of moringa to make
biofuel, fertilizer and livestock feed.(Mahmood, 2011)
Use in crop science
In crop sciences, extract of moringa leaves may be used as seed treatment and foliar application
to boost the growth and productivity. Moringa extracts strengthen plants, and improve resistance
against pests and diseases as enrich with ascorbate, zeatin, minerals, and many other compounds
including essential macro- and microelements, moringa has numerous applications in the field of
agriculture and medicine. Biologist have proved that the defense mechanism against abiotic
stress and growth of plant is promoted by the use of secondary metabolites obtained from this
plant(Hussain & Farooq, 2013).
One of the most important profitable citrus cultivar in Pakistan is Kinnow whose yield and fruit
quality is considerable reduced by the improper nutrient handling. According to modern reports
about growth enhancer properties of M. oleifera, scientists have conducted an experiment to
estimate effectiveness of MLE alone and together with zinc and potassium, on Kinnow. Moringa leaf
extract (MLE) is enriched with phytohormones, phenolics and minerals.MLE application
increased leaf nitrogen, phosphorus, potassium, calcium, manganese, and zinc in both while in
combined application significant lower fruit drop, high yield, fruit weight, phenolics, total
antioxidant, sugar, vitamin C content observed. Moreover MLE foliar application also results in
amplification of SOD and CAT enzyme’s activities. So to increase fruit yield and quality
combined foliar application of MLE, Zn and K can be used.(Nasir et al., 2016)
Maize is a foremost fodder crop that is badly affected by salinity. With the intention to develop
tolerance against the salinity and for the progress of vegetative performance of maize five level
of moringa leaf extract (MLE) i.e. control, 5%, 10%, 15%, and 20% were foliarly applied on
4
maize seedlings. Plants were raised in saline and non-saline hydroponic Hoagland solutions.
According to the results foliar application of moringa leaf extract enhanced the shoot and root
growth considerably and also proved helpful in reducing the Na and increasing the K content of
the leaf. The salt tolerance of maize was improved as the concentration of MLE was increased up
to 15%, but higher level (20%) proved toxic for maize seedling(Ali et al., 2017).
Allelopathy is the advantageous or injurious effects of one plant on another plant by the
discharge of secondary metabolites from plant parts in both natural and farming systems.
Moringa leaf extract (MLE) is usually considered to show progressive or reducing response on
the plant growth in a dose dependent method. To assess the growth affects M.oleifera on the
Cyperus rotundus MLE was applied into pots with 25, 50, 75 and 100% (v/v) concentration
while dist. water used as control. MLE at 100% level especially enhanced the root and shoots
lengths, shoot fresh and dry weights. So M.oleifera enhanced the growth of purple nuts edge yet
at elevated concentrations instead to repress it(Ali et al., 2015)
To explore effect of moringa on growth, yield, physio-biochemical attributes and water use
efficiency (WUH) of maize under drought and full irrigation conditions, a field experiments were
conducted in which MLE was applied as foliar spray alone and together with salicylic acid(SA).
Shortage in irrigation at initial stage results in decrease of all growth parameters, chlorophyll a,
grain yield, increased in carotenoid, proline, membrane permeability and MDA. Overall
combined application of MLE+SA proved helpful to eradicate drought stress as it increase
photosynthetic pigments, growth, proline concentration which is related to MDA accumulation.
Increase in growth yield, grain was also observed over control.(Maswada et al., 2018).
In order to investigate insecticidal activity of moringa, khapra infected sorghum grain were
treated with the moringa powder. Overall results revealed that powder from all parts of this
multi-purpose plant significantly defend the sorghum grains against the larval attack. By the
increase in quantity of powder treatment, increase in rate of larvae mortality was also observed.
However maximum mortality rate of larvae was observed with moringa seed powder treatment
as compared to treatment of moringa flower powder. So M. oleifera powders can be used as
substitute to synthetic insecticides for control of T. granarium.(Ismeal, 2017)
5
Use in phytomedicines
M.oleifera is consider to be supportive for medicinal uses as it contain variety of amino acid,
vitamins, minerals antioxidant carotenoid and compounds having nutraceutical properties (Razis
et al., 2014)In order to evaluate the beneficial properties of moringa in the field of herbal
medicines, experiment was performed by different scientists from all over the world.
Ethanol extracts of moringa seed showed anti-fungal activities in vitro against dermatophytes
such as T. rubrum, T. mentagrophytes, E. Xoccosum, and M. canis. GC–MS analysis of the
chemical composition of the essential oil from leaves showed a total of 44 compounds. Isolated
extracts could be of use for the future development of anti-skin disease agents(Chuang et al.,
2007)
Various bioactive compounds of anticancer properties are known and isolated from moringa.
One of those compound is, niazimicin that exhibits anticancer activity against EB virus
(Murakami et al., 1998) Moreover further study showed that moringa seeds have potential to
affect those enzymes which are involved in metabolism of hepatic carcinogen(Parvathy &
Umamaheshwari, 2007; Budda et al., 2011).
Leaf extract of moringa contain different alkaloid and polyphenols including moringinine, rutin,
Quercitin-3-glycoide, kaempferol and many others which are evidence of its antidiabetic, cardiac
stimulant properties. Besides these moringa leaf extract also involve in lowering of body weight,
blood cholesterol, serum triglycerides. Experiment also showed its hypolipidemic properties (Ara
et al., 2008)((Mittal et al., 2007; Ezejindu et al., 2013) .
Experiments showed hepatoprotective properties of moringa. Flower extract of moringa contain

Quercitin which ensure considerable protection liver damage induced by CCl4.On the other hand
seed extract of moringa control CCl4 thus leads to the protection against liver fibrosis (Hamza,
2010).
In case of eye disease treatment leaves, utilization of different part of this incredible tree found
very helpful. Experiments have proved that intake of moringa leaves and pods results in
improvement of vitamin A. Besides this it also delayed the development of cataract.
6
The efficacy of seed kernels of moringa to reduction in bronchial asthma and progress of
coexisting respiratory functions was also reported in literature(Nambiar et al., 2003; Agrawal &
Mehta, 2008). According to study moringa seed are found to be helpful in protecting animals
from the poisonous effects of arsenic which can enter their body either from drinking water,
inhaled or absorbed by the skin(Gupta et al., 2005).
Use in biofuel production and water coagulation treatment
Day by day increase in fuel demand has leaded the scientists to the production of biofuel from
non edible oil sources to reduce expenditure of fossil fuel import especially for developing
countries. Biodiesel production from M.oleifera needs only a single step trans esterification
process. Moringa extracted biodiesel and diesel blend was when tested on four stroke engine it
give increase in brake specific fuel consumption and decrease in CO2 and oxide of nitrogen as
compared to ordinary diesel. Results reveals the potential of this tree to be used for biodiesel
production which can be used as alternate fuel without needing any modification in engine.
(Zeeshan et al., 2016)
In an experiment crude water extract of M.oleifera seeds was used to treat industrial and
municipal wastewater in comparison with alum. According to results moringa seed extract
effectively removed suspended solid, metals and micro organisms and produce less sludge
though nutrient and COD was not removed successfully as compared to alum. It is proved that
moringa is quite effective to be used as primary coagulant but for improved outcome purified
protein from moringa should be used(Ndabigengesere & Narasiah, 1998).
Use in livestock and poultry feed
In an experiment moringa leaves were used to feed dairy cows in order to test their effect on dry
mater intake, digestibility, milk production and its composition. The results showed that the
inclusion of Moringa as a protein supplement to low quality diets improved dry matter intake,
digestibility of the diet and increased milk production with no effect on milk
composition.(Sánchez et al., 2006).
Moringa based livestock meal is a cheap source of protein as compared to commercially

available concentrate. To examine effect of moringa leaf meal, soy bean concentrate and
7
commercially available concentrate on the milk yield and it composition an experiment was
performed in which dairy cow were fed with elephant grass with 20% soybean concentrate,20%
moringa leaf concentrate and elephant grass and readymade protein concentrate(having the same
energy and protein content as moringa leaf concentrate).Result showed that milk production and
composition were not significantly changed variations among treatments. Thus locally
prepared Moringa leaf meal can effectively substitute the ingredients available in market to
make concentrate for dairy cows with the same protein and energy levels.(Araica et al., 2011)
Moringa leaves used by the scientists to broiler hens and chicken to improve their health, growth,
digestibility of dry matter, meat weight. It has been proved that use of Moringa oleifera leaf meal
in combination with soybeans as protein source produce broiler of same weight and growth rate
as compared to those broiler fed with expensive commercial feed. Moreover studies have also
revealed the use of MOLM as low cost substitute poultry feed in comparison with other poultry
feeds (Gadzirayi et al., 2012; Molepo, 2014)
Aims and Objectives
As far as aims and objectives are concerned, the study reveals the bio chemical, medicinal,
industrial, poultry and live stock, nutritional, pharmaceutical importance of plant. It also
elucidates the variation in biochemical and morphological parameters in different edaphic
settings of Southern Punjab. This plant also shows the property of alternate food source as it
contains all the essential elements to fulfill the nutritional requirements for malnourished
persons.
8
Chapter # 2 Material and method
Site description
Plant and soil samples were collected from different region of southern Punjab, Pakistan
including Multan, Shujabad, and Muzafergerh.
Sites coordinates Temperature Annual rainfall

Shujabad 29o52’45” N 44oC 157mm
71o18’10”E 3oC(lowest)
Multan 30o11’52”N 52oC(highest) 186mm
71o28’11”E -1oC(lowest)
Muzafergerh 30o4’10”N 54oC(highest) 127mm

71o11’39”E -1oC(lowest)
9
Morphological Parameters
Branch length (cm)
Branch length was measured with a centimeter ruler from selected sample of plant.
Leaf fresh weight (g)
Leaf fresh weight was calculated with the help of digital balance (chyo MK-200B).
Leaf dry weight (g)
Samples were oven dried at 70oC and their dry weight was determined with the help of digital
balance (chyo MK-200B).
Leaf width
Leaf width was measured by centimeter ruler.
Leaf length
Leaf length was measured by centimeter ruler.
10
Biochemical Estimation
Chlorophyll and Carotenoids estimation
0.2g fresh leaf sample was grinded in 2ml of 80% of acetone and raised the final vol. upto 10ml
then the mixture was filtered carefully. The samples were kept at 4˚C overnight and their
readings were taken at 663 nm, 645 nm and 480 nm with the help of spectrophotometer
according to protocol(Arnon, 1949)
Ascorbic acid estimation
0.25g fresh leaf samples were mixed with 5 mL solution of 6% trichloroacetic acid and then
filtered it.2ml filtrate was mixed with 1.0 mL of 2% 2, 4 dinitrophenylhydrazine (2, 4 - DNPH)
and then with one drop of 10% thiourea. The samples were incubated in a water bath at 100˚C
for 15 min, cooled at room temperature and added 2.5 mL of 80% H2SO4 to each sample. The
optical density of each sample was noted at 530 nm using a spectrophotometer(Mukherjee &
Choudhuri, 1983)
Protein estimation
2.5g of fresh leaves were grinded in 1ml of sodium phosphate buffer (pH 7) to fine paste, volume
raised up to 4 ml in folkin tubes. The mixture was then centrifuge for 15 minute at 4000 rpm at
room temperature. The supernatant was picked with the help of micropipette for further use and
pallet was discarded.50µl of supernatant and 2.5ml of reagent was mixed and after half an hour
optical density was observed at 595nm with the help of spectrophotometer(Bradford, 1976)
Total soluble protein (mg/g fresh wt.) =reading of sample× vol. of the sample ×dil. Factor
Fresh wt. of sample× 1000
Amino acid content estimation
0.2g of fresh leaves were grinded in 2ml sodium phosphate buffer (pH 7) and final vol. raised up
to 4ml, the mixture was centrifuged for 15 mints at 4000 rpm.1ml of 10% pyridine and 1ml of
2% ninhydrin solution was added to the 1ml supernatant then the mixture was heated in the water
11
bath at 90o C for 30 minutes and cooled down. Upto 50ml of dist. water was used to make
dilution of each sample and the optical density was observed at 570nm with the help of
spectrophotometer(Hamilton, 1943).
Totle free aminoacid= reading× vol. of extract× dilution factor
Wt. of sample× 1000
Digestion mixture
Add 0.42g of Se and 14g of LiSO4 . 2H2O to 350ml of 100 vol. H2O2 .Mix well and add
420mlH2SO4 carefully in ice bath to avoid explosion.Stor the mixture at 2oC.
Ion analysis
0.1g of dry leaf samples were added in 2ml of digestion mixture and sample were kept overnight
at room temperature, next day place them at hot plate for digestion at moderate temperature.
After half an hour 0.5ml perchloric acid was added to decolorize the mixture,continue the
digestion till mixture become colorless. The sample were diluted by adding 50ml of dist. water
and filtered for further use. With the help of flam photometer(Digiflame 2000) Na+ and K+
concentration was measured by spotting emission through known standards with samples
according to protocol(Munns et al., 2012).
Total elemental %= C*(mg)× soln. vol(ml)
10× aliquot (ml) × sample wt.(g)
C*=mg element obtained from caliberation curve
12
Soil Analysis
Soil samples of selected sites were also examined with the cooperation of “Engro Soil and
Water Teting Lab”.Soil analysis include measument of pH,EC and SAR.
pH Measurment
The protocol(Mckeague, 1978) followed for the estimation of soil pH in 1:1 soil:water
suspention. 10g air dried soil I weighted in 25 mL plastic beaker and 25 mL distilled water was
added in it. The mixture was left for 1 hour to allow most of the sediment to settle from soil:
water suspension. The pH of supernatant was measured with the help of pH meter.
Electrical conductivity Measurment
5 g air-dry soil was added in 50 mL dist. water by using a graduated cylinder and kept that
mixture overnight. Next moring mixuter was filtered and electrical conductivity of filtrate was
measured by EC meter. Before meauring the EC of filtrate meter was caliberated by the
standrized solution of KCl.(Estefan et al., 2013)
SAR Determination
5 g air-dry soil was added in 50 mL dist. water by using a graduated cylinder and kept that
mixture for 16 hrs. After that mixture was filtered through filter paper in conical flask. Run the
flame photometer at least half hour to warm up the apparatus.Calibrate the apparatus with
standard solution of KCl 25 ppm. Feed the filtrate on flame photometer and note the reading of
sodium by adjusting the sodium filter on flame photometer.(Bohn et al., 1985)
13
Chapter # 3 Results
To study the morpho and biochemical attributes of Moringa oleifera across edaphically different
habitats, soil and plant samples were collected from different sites and several tests were
performed including estimation of chlorophyll, carotenoid, ascorbic acid, protein, amino acid,
ions analysis along with morphological parameters.
Leaves fresh weight
Results for the measurement of leave fresh weight of Moringa oleifera are given as mean values
in fig 1and table 3.1a.
Mean values data shows that samples collected from different sites exhibit variation in fresh
weight and variability found to be significant. M.oleifera collected from Multan showed highest
value for the parameter while the M.oleifera collected from Shujabad showed lowest value for
the parameter.
Analysis of variance for fresh weight is given in table 3.1b.ANOVA showed that plants replicate
from same sites did not differ significantly while from diverse site differ significantly.
14
10
9
8
leaves fresh wt. (g)

7
6
5
4
3
2
1
0
shujabad multan muzafergerh
M.oleifera
Fig 1: Estimation of leaves fresh weight (g) M.oleifera under differential edaphic site of southern
Punjab
15
Table 3.1a: Mean, S.D, S.E table of leaves fresh weight (g) of M.oleifera under differential
edaphic sites of southern Punjab.
Sites Mean S.D S.E

Shujabad 3.96 ± 0.51439 ±0.25720
Multan 8.905 ± 0.43562 ± 0.21781
Muzafergerh 5.815 ± 0.42273 ± 0.21136
Table 3.1b: Analysis of variance leaves fresh weight (g) of M.oleifera under differential edaphic
sites of southern Punjab.
Source of SS Df MS F F crit.
variance
Plant 0.0257 3 0.00867 0.01122NS 3.862548
Sites 91.587 3 30.529 39.9740*** 3.862548
Residual 6.8735 9 0.76372
Total 98.4862 15
NS = Non-significant
*** = highly significant
16
Leaves dry weight
Results for the measurement of leave dry weight of Moringa oleifera are given as mean values in
fig 2and table 3.2a.
Mean values data shows that samples collected from different sites exhibit non significant
variation in dry weight. M.oleifera collected from Multan and Muzafergerh showed similar value
for the parameter while the M.oleifera collected from Shujabad showed lowest value for the
parameter.
Analysis of variance for dry weight is given in table 3.2b.ANOVA showed non significant
variance for the parameter.
17
3
2.5
leaves dry wt. (g)
1.5
0.5
0
M.oleifera
Fig 2: Estimation of leaves dry weight (g) M.oleifera under differential edaphic site of southern
Punjab
18
Table3.2a: Mean, S.D, S.E table of leaves dry weight (g) of M.oleifera under differential edaphic
Sites Mean S.D S.E

Shujabad 1.1225 ± 0.14569 ±0.07284
Multan 2.3175 ± 0.22618 ± 0.11309
Muzafergerh 2.235 ± 0.18930 ± 0.09465
Table 3.2b: Analysis of variance leaves dry weight (g) of M.oleifera under differential edaphic
variance
plant 0.68973 3 0.22991 0.44643 NS 3.86255
Sites 4.67353 3 1.55784 3.02498 NS 3.86255
residual 4.63493 9 0.51499
Total 9.99818 15
NS = non significant
19
Leaf length
Results for the measurement of leaf length of Moringa oleifera are given as mean values in Fig
3and Table 3.3a.
Mean values data shows that samples collected from different sites exhibit variation in leaf
length and variability found to be significant. M.oleifera collected from Multan showed highest
value for the parameter while the M.oleifera collected from Shujabad showed lowest value for
the parameter.
Analysis of variance for leaf length is given in Table 3.3b.ANOVA showed that plant replicate
20
60
50
40
leaf length(cm) 30
20
10
0
M.oelifera
Fig3: Estimation of leaf length (cm) of M.oleifera under differential edaphic site of southern
Punjab
21
Table3.3a: Mean, S.D, S.E table of leaf length (cm) of M.oleifera under differential edaphic sites
of southern Punjab.
Sites Mean S.D S.E

Shujabad 35.6 ± 4.457952 ±2.228976
Multan 50.375 ± 2.926175 ± 1.463087
Muzafergerh 43.875 ± 3.275541 ± 1.63777
Table 3.3b:Analysis of variance leaf length (cm) of M.oleifera under differential edaphic sites of
southern Punjab.
variance
Plant 52.3425 3 17.4475 2.238374NS 3.862548
Sites 5428.543 3 1809.514 232.1461*** 3.862548
Residual 70.1525 9 7.794722
Total 5551.038 15
NS= Non significant *** = highly significant
22
Leaf width
Results for the measurement of leaf width of Moringa oleifera are given as mean values in fig
4and table 3.4a.
Mean values data shows that samples collected from different sites exhibit variation in leaf width
and variability found to be significant. M.oleifera collected from Multan showed highest value
for the parameter while the M.oleifera collected from Shujabad showed lowest value for the
parameter.
Analysis of variance for leaf width is given in table 3.4b.ANOVA showed that plant replicates
23
42
41
40
leaf width (cm) 39
38
37
36
35
M.oleifera
Fig 4: Estimation of leaf width (cm) of M.oleifera under differential edaphic site of southern
Punjab
24
Table3.4a: Mean, S.D, S.E table of leaf width (cm) of M.oleifera under differential edaphic sites
of southern Punjab.
Sites Mean S.D S.E

Shujabad 38 ± 1.08012 ±0.54006
Multan 40.6 ± 3.22594 ± 1.61297
Muzafergerh 39.075 ± 2.42676 ± 1.21338
Table 3.4 b: Analysis of variance leaf width (cm) of M.oleifera under differential edaphic sites of
southern Punjab.
variance
Plant 8.65688 3 2.88563 0.53294NS 3.862548
Sites 4059.832 3 1353.277 249.9351*** 3.862548
Residual 48.73063 9 5.414514
Total 4117.219 15
25
Chlorophyll estimation
Results for the estimation of chlorophyll a content in Moringa oleifera are given as mean values
Mean values data shows that samples collected from different sites exhibit variation in
chlorophyll content and variability found to be significant. M.oleifera collected from Shujabad
showed highest value for the parameter while the M.oleifera collected from Muzafergerh showed
lowest value for the parameter.
Analysis of variance for chlorophyll a is given in table 3.5b.ANOVA showed that plant
replicates from same sites did not differ significantly while from diverse site differ significantly.
26
0.7
0.6
0.5
Chlorophyll a (mg/g)
0.4
0.3
0.2
0.1
0
Shujabad Multan Muzafergerh
M. oleifera
Fig 5: Estimation of chlorophyll a (mg/g) from M.oleifera collected from different sites i.e.
Shujabad, Multan, and Muzafergerh across southern Punjab.
27
Table3.5a: Mean, S.D, S.E table of chlorophyll a (mg/g fwt) in leaves of M.oleifera under
differential edaphic sites of southern Punjab.
Sites Mean S.D S.E

Shujabad 0.58306 ± 0.03999 ±0.02000
Multan 0.50471 ± 0.14297 ± 0.07148
Muzafergerh 0.35087 ± 0.10928 ± 0.07148
Table3.5b: Analysis of variance of chlorophyll a (mg/g) in leaves of Moringa oleifera under

differential edaphic sites of southern Punjab
variance
Plants 1.390751 3 0.463584 1.124235NS 3.862548
Sites 12.35829 3 4.119429 3.862548

9.990017***
Residual 3.711191 9 0.412355
Total 17.46023 15
NS= non significant
***= highly significant
28
Estimation of Chlorophyll b
Results for the estimation of chlorophyll b content in Moringa oleifera are given as mean values
showed highest value for chlorophyll b while the M.oleifera collected from Muzafergerh showed
Analysis of variance for chlorophyll b is given in table 3.6b.ANOVA showed that plant
29
0.35
0.3
chlorophyll b (mg/g)
0.25
0.2
0.15
0.1
0.05
0
M. oleifera
Fig 6: Estimation of chlorophyll b (mg/g) from M.oleifera collected from different sites i.e.
30
Table 3.6a: Mean, S.D, S.E table of chlorophyll b (mg/g fwt) in leaves of M.oleifera under
Sites Mean S.D S.E

Shujabad 0.28676 ± 0.09863 ±0.04932
Multan 0.19194 ± 0.07541 ±0.03770
Muzafergerh 0.12747 ±0.04073 ± 0.02037
Table 3.6b: Analysis of variance of chlorophyll b (mg/g) in leaves of Moringa oleifera under
Source of SS df MS F F crit.
variance
Plants 1.513055 3 0.504352 1.282914NS 3.862548
Sites 15.89298 3 5.297661 13.4756*** 3.862548
residual 3.538168 9 0.39313
Total 20.94421 15
NA = non significant
31
Estimation of chlorophyll a/b
Results for the estimation of chlorophyll a/b content in M. oleifera are given as mean values in
fig 7and table 3.7a.
chlorophyll a/b content. M.oleifera collected from Shujabad showed highest value for
chlorophyll b while the M.oleifera collected from Muzafergerh showed lowest value for the
parameter.
Analysis of variance for chlorophyll a/b is given in table 3.6b.ANOVA showed that non
significant variance for chlorophyll a/b.
32
3.5
2.5
chlorophyll a/b (mg/g)
1.5
0.5
0
M. oleifera
Fig 7: Estimation of chlorophyll a/b (mg/g) from M.oleifera collected from different sites i.e.
Shujabad, Multan, and Muzafergerh across southern Punjab
33
Table 3.7a: Mean, S.D, S.E table of chlorophyll a/b (mg/g fwt) in leaves of M.oleifera under
Sites Mean S.D S.E

Shujabad 2.20763 ± 0.67607 ±0.33803
Multan 2.72975 ± 0.35165 ± 0.17582
Muzafergerh 2.77629 ± 0.29738 ± 0.14869
Table 3.7b: Analysis of variance of Chlorophyll a/b (mg/g) in leaves of M. oleifera under
variance
Plants 1.093423 3 0.364474 0.554656 NS 3.862548
Sites 0.812748 3 0.270916 0.412279 NS 3.862548
residual 5.914056 9 0.657117
Total 7.820226 15
34
Estimation of total chlorophyll (a+b)
Results for the estimation of total chlorophyll (a+b) content in M. oleifera are given as mean
values in fig 8and table 3.8a.
Mean values data shows that samples collected from different sites exhibit variation in total
showed highest value for chlorophyll b while the M.oleifera collected from Muzafergerh showed
Analysis of variance for total chlorophyll is given in table 3.8b.ANOVA showed that plant
35
1
0.9
total chlorophyll ( a+b) (mg/g) 0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
M.oleifera
Fig 8: Estimation of total chlorophyll (a+b) (mg/g) from M.oleifera collected from different sites
i.e. Shujabad, Multan, and Muzafergerh across southern Punjab.
36
Table 3.8a: Mean, S.D, S.E table of total chlorophyll (a+b) (mg/g fwt) in leaves of M.oleifera
under differential edaphic sites of southern Punjab.
Sites Mean S.D S.E

Shujabad 0.87258 ± 0.06584 ±0.03292
Multan 0.69908 ± 0.21715 ± 0.10857
Muzafergerh 0.48003 ± 0.10857 ± 0.07456
Table 3.8b: Analysis of variance of total chlorophyll (a +b) (mg/g) in leaves of M. oleifera under
variance
Plants 1.711813 3 0.570604 1.463354 NS 3.862548
Sites 10.20425 3 3.401416 8.723165*** 3.862548
residual 3.509362 9 0.389929
Total 15.42542 15
37
Estimation of carotenoid
Results for the estimation of carotenoid content in M. oleifera are given as mean values in fig
9and table 3.9a.
Mean values data shows that samples collected from different sites exhibit variation in total
carotenoid content and variability found to be significant. M.oleifera collected from Shujabad
showed highest value for carotenoid while the M.oleifera collected from Muzafergerh showed
Analysis of variance for leaf length is given in table 3.9b.ANOVA showed non significant
variance for carotenoid.
38
2.5
2
carotenoid (mg/g)
1.5
0.5
0
M. oleifera
Fig 9: Estimation of carotenoids (mg/g) from M.oleifera collected from different sites i.e.
39
Table 3.9a: Mean, S.D, S.E table of carotenoids (mg/g fwt) in leaves of M.oleifera under
Sites Mean S.D S.E

Shujabad 2.17895 ±0.11273 ±0.05637
Multan 1.77183 ± 0.51861 ± 0.25930
Muzafergerh 1.34026 ± 0.38165 ± 0.19082
Table 3.9b: Analysis of variance of carotenoids (mg/g) in leaves of M. oleifera under differential
edaphic sites of southern Punjab
variance
Plants 2.52685 3 0.84228 2.01873 NS 3.862548
Sites 3.03372 3 1.01124 2.42368 NS 3.862548
residual 3.75510 9 0.41723
Total 9.31567 15
40
Estimation of chlorophyll/carotenoid
Results for the estimation of chlorophyll/carotenoid content in M. oleifera are given as mean
values in fig 9and table 3.9a.
chlorophyll/carotenoid content and variability found to be significant. M.oleifera collected from
Multan showed highest value for chlorophyll/carotenoid while the M.oleifera collected from
Muzafergerh showed lowest value for the parameter.
Analysis of variance for chlorophyll/carotenoid is given in table 3.9b.ANOVA showed that plant
41
0.7
0.6
chlorophyll/carotenoid (mg/g)
0.5
0.4
0.3
0.2
0.1
0
M. oleifera
Fig 9: Estimation of carotenoids/chlorophyll from M.oleifera collected from different sites i.e.
42
Table 3.9a: Mean, S.D, S.E table of chlorophyll/carotenoid (mg/g fwt) in leaves of M.oleifera
under differential edaphic sites of southern Punjab.
Sites Mean S.D S.E

Shujabad 0.40020 ± 0.01367 ±0.00683
Multan 0.61776 ±0.44408 ± 0.22204
Muzafergerh 0.35648 ± 0.00896 ±0.00448
Table 3.9b: Analysis of variance of chlorophyll/carotenoids (mg/g) in leaves of M. oleifera under

variance
Plants 1.21570 3 0.40523 0.83329 NS 3.862548
Sites 12.6642 3 4.22139 8.68057 *** 3.862548
residual 4.37674 9 0.48630
Total 18.2566 15
43
Estimation of protein
Results for the estimation of protein content in M. oleifera are given as mean values in fig 10 and
table 3.10a.
Mean values data shows that samples collected from different sites exhibit variation in protein
content and variability found to be significant. M.oleifera collected from Multan showed highest
value for protein while the M.oleifera collected from Muzafergerh showed lowest value for the
parameter.
Analysis of variance for protein is given in table 3.10b.ANOVA showed that plant replicates
from same sites did not differ significantly while from diverse site differ significantly
44
35
30
25
protein (µg/g fwt)
20
15
10
0
Shujahbad Multan Muzafrgerh
M. oleifera
Fig 10: Estimation of total soluble proteins µg/g from fresh leaves of M.oleifera under
45
Table: Mean, S.D, S.E table of Protein (µg/g fwt) in leaves of M.oleifera under differential
Sites Mean S.D S.E

Shujabad 26.08567 ± 0.572431 ±0.286216
Multan 28.97759 ± 6.25762 ± 3.12881
Muzafergerh 22.20307 ± 1.523474 ± 0.761737
Table 3.10b: Analysis of variance of Protein (µg/g fwt) in leaves of M. oleifera under differential
variance
Plants 26.08092 3 8.69364 0.749894 NS 3.862548
Sites 1714.89 3 571.6299 49.30751 *** 3.862548
residual 104.3385 9 11.59316
Total 1845.309 15
46
Estimation of amino acid
Results for the estimation of amino acid content in M. oleifera are given as mean values in fig 11
and table 3.11a.
Mean values data shows that samples collected from different sites exhibit variation in amino
acid content and variability found to be significant. M.oleifera collected from Multan showed
highest value for protein while the M.oleifera collected from Muzafergerh showed lowest value
for the parameter.
Analysis of variance for amino acid is given in table 3.11b.ANOVA showed that plant replicates
from same sites did not differ significantly while from diverse site differ significantly
47
12
10
Amino acid µg/g fwt
0
Shujahbad Multan Muzzafargarh
M. oleifera
Fig 11: Estimation of amino acid (µg/g) from fresh leaves of M.oleifera under differential
48
Table 3.11a: Mean, S.D, S.E table of amino acid (µg/g fwt) in leaves of M.oleifera under
Sites Mean S.D S.E

Shujabad ± 9.942016 ±0.886222
7.259
Multan 1.6395 ± 1.772445 ± 1.148106
Muzafergerh 0.819783 ± 0.886222 ± 0.574053
Table 3.11b: Analysis of variance of amino acid (µg/g) in leaves of M. oleifera under differential
variance
plant 1.594269 3 0.531423 3.862548
0.192472 NS
sites 115.0183 3 38.33945 3.862548
13.88586 ***
residual 24.84937 9 2.761042
Total 141.462 15
49
Estimation of ascorbic acid
Results for the estimation of ascorbic acid content in M. oleifera are given as mean values in fig
12 and table 3.12a.
Mean values data shows that samples collected from different sites exhibit variation in ascorbic
acid content and variability found to be significant. M.oleifera collected from Multan and
Muzafergerh showed highest value for parameter while the M.oleifera collected from Shujabad
showed lowest value for the parameter.
Analysis of variance for ascorbic acid is given in table 3.12b.ANOVA showed that plant
50
120
100
Ascorbic acid (µg/g fwt)
80
60
40
20
0
shujabad multan muzafergrh
M. oleifera
Fig 12: Estimation of amino acid (µg/g) from fresh leaves of M.oleifera under differential
51
Table 3.12a: Mean, S.D, S.E table of ascorbic acid (µg/g fwt) in leaves of M.oleifera under
Sites Mean S.D S.E

Shujabad 54.112 ± 4.205093 ±2.102546
Multan 110.653 ± 2.690805 ± 1.345402
Muzafergerh 109.442 ± 5.166866 ± 2.583433
Table 3.12b: Analysis of variance of ascorbic acid (µmg/g) in leaves of M. oleifera under
variance
sites 85.28096 3 28.42699 3.862548
3.430528 NS
Ascorbic acid 32057.22 3 10685.74 3.862548
1289.54 ***
residual 74.57827 9 8.286475
Total 32217.08 15
52
Estimation of Na ions
Results for the estimation of Na ions in M. oleifera are given as mean values in fig 13 and table
3.13a.
Mean values data shows that samples collected from different sites exhibit variation in Na ions
content and variability found to be significant. M.oleifera collected from Multan showed highest
value for parameter while the M.oleifera collected from Shujabad showed lowest value for the
parameter.
Analysis of variance for Na ion content is given in table 3.13b.ANOVA showed that plant
replicates from same sites did not differ significantly while from diverse site differ significantly
53
18
16
14
12
Na ions (mg/g)
10
8
6
4
2
0
M.oleifera
Fig 13: Estimation of Na ion (mg/g) from dry leaves of M.oleifera under differential edaphic
sites of southern Punjab
54
Table 3.13a: Mean, S.D, S.E table of Na ions (mg/g) in leaves of M.oleifera across three
different habitat of Southern Punjab.
Sites Mean S.D S.E

Shujabad 9.226031 ± 0.78114 ±0.39057
Multan 15.47765 ± 0.254465 ± 0.127232
Muzafergerh 10.09164 ± 0.759796 ± 0.379898
Table 3.13b: Analysis of variance Na ions (mg/g) in leaves of M. oleifera under differential
variance
sites 3.725309 3 1.24177 2.221256 NS 3.862548
Na ion 340.1328 3 113.3776 202.8078 *** 3.862548
residual 5.031356 9 0.55904
Total 348.8894 15
55
Estimation of K ions
Results for the estimation of K ions in M. oleifera are given as mean values in fig 14 and table
3.14a.
Mean values data shows that samples collected from different sites exhibit variation in K ions
content and variability found to be significant. M.oleifera collected from Muzafergerh showed
highest value for parameter while the M.oleifera collected from Shujabad showed lowest value
for the parameter.
Analysis of variance for K ion content is given in table 3.14b.ANOVA showed that plant
56
70
60
50
K ions (mg/g)
40
30
20
10
0
M.oleifera
Fig14: Estimation of K ions (mg/g) from dry leaves of M.oleifera under differential edaphic sites
of southern Punjab.
57
Table3.14a: Mean, S.D, S.E table of K ions (mg/g) in leaves of M.oleifera across three different
habitat of Southern Punjab.
Sites Mean S.D S.E

Shujabad 43.43003 ± 2.20163 ± 1.100815
Multan 52.03802 ± 2.766983 ± 1.3834
Muzafergerh 61.55971 ± 1.893686 ± 0.946843
Table3.14b: Analysis of variance K ions (mg/g) in leaves of M. oleifera under differential

Source of SS df MS F F crit.
variance
sites 2.563672 3 0.854557 0.151683 NS 3.862548
K ions 8028.074 3 2676.025 474.9909 *** 3.862548
residual 50.70459 9 5.633844
Total 8081.342 15
58
Estimation of soil pH
Results for the estimation of pH from soil samples are given as mean values in fig 15 and table
3.15a.
Mean values data shows that samples collected from different sites exhibit variation in pH vales
and variability found to be significant. Soil samples collected from Shujabad showed highest
value for parameter while the soil sample collected from Multan and Muzafergerh showed
Analysis of variance for estimation of pH is given in table 3.15b.ANOVA showed that plant
59
8.05
7.95
7.9
soil pH
7.85
7.8
7.75
7.7
7.65
7.6
Fig15: Estimation of pH of soil samples under differential edaphic sites of southern Punjab
60
Table 3.15a: Mean, S.D, S.E table of pH of soil samples under differential edaphic sites of
southern Punjab.
Sites Mean S.D S.E

Shujabad 8 ± 0.05 ±0.025
Multan ± 0.05 ± 0.025

7.8
Muzafergerh 7.8 ± 0.05 ± 0.025
Table3.15b: Analysis of variance of soil pH of soil samples under differential edaphic sites of
southern Punjab
variance
replicates 1.28188 3 0.42729 1.02807 NS 3.862548
Sites 85.7069 3 28.56896 68.73734 *** 3.862548
residual 3.74062 9 0.41562
Total 90.72938 15
61
Estimation of electrical conductivity
Results for the estimation of EC from soil samples are given as mean values in fig 16 and table
3.16a.
Mean values data shows that samples collected from different sites exhibit variation in EC vales
62
70
60
50
EC (dS/m)
40
30
20
10
0
shujabad multan muzafargarh
Fig16: Estimation of EC of soil samples under differential edaphic sites of southern Punjab
63
Table 3.16a: Mean, S.D, S.E table of electrical conductivity (dS/m) of soil under differential
Sites Mean S.D S.E

Shujabad 60.125 ± 0.0957 ±0.047871
Multan 2.0925 ± 0.005 ± 0.0025
Muzafergerh 1.7225 ± 0.005 ± 0.0025
Table3.16b: Analysis of variance of electrical conductivity (dS/m) of soil samples under

variance
replicates 1.2757 3 0.42523 1.0200 NS 3.86255
Sites 10100.17 3 3366.724 8075.937 *** 3.86255
residual 3.75195 9 0.416883
Total 10105.2 15
64
Estimation of available Potash
Results for the estimation of available potash (K) from soil samples are given as mean values in
fig 17 and table 3.17a.
Mean values data shows that samples collected from different sites exhibit variation in K vales
65
1200
1000
availble potash K (ppm)
800
600
400
200
0
shujabad multan muzafrgarh
soil
Fig 3.17: Estimation of available K in soil samples under differential edaphic sites of southern
Punjab.
66
Table3.17a: Mean, S.D, S.E table of available potash K(ppm) in soil samples under differential
Sites Mean S.D S.E

Shujabad 1000 ± 0.613 ±0
Multan 125 ± 0.47 ± 0.23583
Muzafergerh 125 ± 0.5 ± 0.25
Table 3.17b: Analysis of variance available potash K (ppm) in soil samples under differential
variance
replicates 1.5 3 0.5 0.9 NS 3.862548
Sites 2557022 3 852340.5 1534213 *** 3.862548
residual 5 9 0.55556
Total 2557028 15
67
Estimation of SAR
Results for the estimation of SAR from soil samples are given as mean values in fig 18 and table
3.18a.
Mean values data shows that samples collected from different sites exhibit variation in SAR
vales and variability found to be significant. Soil samples collected from Shujabad showed
highest value for parameter while the soil sample collected from Muzafergerh showed lowest
value for the parameter.
Analysis of variance for estimation of SAR is given in table 3.18b.ANOVA showed that plant
68
12
10
8
SAR
0
shujabad multan muzafargarh
Soil
Fig 18: Estimation of SAR in soil samples under differential edaphic sites of southern Punjab.
69
Table 3.18a: Mean, S.D, S.E table of SAR in soil samples under differential edaphic sites of
southern Punjab.
Sites Mean S.D S.E

Shujabad 10.25 ± 0.25166 ±0.12583
Multan 2.825 ± 0.09574 ± 0.04787
Muzafergerh 2.375 ± 0.18930 ± 0.09465
Table 3.18b: Analysis of variance of SAR in soil samples under differential edaphic sites of
southern Punjab
variance
replicates 0.8675 3 0.289167 0.583847 NS 3.862548
Sites 177.5325 3 59.1775 119.4835 *** 3.862548
residual 4.4575 9 0.495278
Total 182.8575 15
70
Chapter # 4 Discussion
Increase in leaf fresh and dry weight is observed in Multan. It shows maximum biomass
production is related to this particular habitat as reported by other authors increase in different
crops (Poorter & Nagel, 2000; Ogawa et al., 2015). Leaf length is observed to be increase in
Multan region which showed increase rate of photosynthesis by increase in leaf blade in this.
Leaf width also showed the same results which again indicate the increase in leaf surface area,
which increase in stomata content hence favours more photosynthesis.
Chlorophyll a content seen to be maximum in Shujabad region which shows maximum rate of
photosynthesis and increase photosystem for light capturance. Chlorophyll b also showed the
same results. Carotenoid is also observed maximum in Shujabad which shows this area is better
in photosynthetic and bio mass production. Basic function of carotenoid is protection of
oxidative damage to plants during photosynthesis, act as antioxidant and also helpful in plant
development(Shumbe et al., 2014; Nisar et al., 2015)
High value of ascorbic acid concentration is observed in Multan and Muzafergerh. Ascorbic
acid is an anti oxidant whose concentration increase is related to salt tolerance as reported in
different crops (Saleem et al., 2012; Shaheen et al., 2013).
Soluble protein and total free amino acid contents are observed maximum in Multan which is
related to stress tolerance in plants and shows increase in photosynthesis.(Amin et al., 2009;
Zonouri et al., 2014)
Increased value of Na ions observed in Multan. Experiment have shown that increase in Na ions
is related to salt stress (Bernstein, 1975; Ali et al., 2017) Increased value of K ions observed in
Muzafergerh. High value for pH, SAR, EC, available potash is observed for Shujabad. The
increase in K ion uptake is related to presence such of antioxidant and growth regulators that
help to plant to recover from stress(Cakmak, 2005; Yasmeen et al., 2013).
According to the criteria for salt effected soil (Bohn et al., 1985), samples from Muzafergerh
and multan shows the value of pH, EC and SAR in the range reveling that sample from selected
site of these two habitat are not affected by salinity. On the other hand pH and SAR results of
Shujabad habitat exhibit that this soil is not affected by the salinity with the exception of
showing highest EC value as compared to two other habitat. Results from available potash shows
71
that sample from Shujabad have highest value for the parameter as compared to other selected
habitat, which reveals the Shujabad soil sample as satisfactory fertility according to criteria. EC
value is directly related to salt and ion concentration present in the soil. Soil with higher values
of soluble ions i.e. C, Ca, Mg, K show higher EC value.(Miranda et al., 2006), moreover higher
EC value may related to the capacity to retain salt and ions of soil rich in organic matter and clay
particle(Carmo et al., 2016)
72
Conclusion
To study morpho and biochemical attribute of M. oleifera under differential edaphic habitats
samples were collected from different sites of Southern Punjab. Analysis of different parameters
showed that increase in leaf width, leaf length, fresh weight and dry weight is related to
increased biomass production and rate of photosynthesis. Biochemical analysis of leave samples
showed that edaphic properties of Shujabad habitat indicate better growth rate as compared to
other habitats though the difference of growth rate in other two habitat i.e. Multan and
Muzafergerh was not so poor indicating that this plant can survive over the range of habitat with
no significant differences in biochemical composition but the exception was present in case of
ionic concentration which show the influence of soil properties on biochemical properties of M.
oleifera.
73
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Morpho-Biochemical attributes of Moringa oleifera

Lam. of edaphically differential settings

A thesis submitted in partial fulfillment of the requirement for the degree

INSTITUTE OF PURE AND APPLIED BIOLOGY

BAHAUDDIN ZAKARIYA UNIVERSITY MULTAN

(Prof. Dr. Seema Mahmood)

(Prof .Dr.Muhammad Naeem)

Use in crop science

Experiments showed hepatoprotective properties of moringa. Flower extract of moringa contain

Use in biofuel production and water coagulation treatment

Use in livestock and poultry feed

Moringa based livestock meal is a cheap source of protein as compared to commercially

Aims and Objectives

Sites coordinates Temperature Annual rainfall

Muzafergerh 30o4’10”N 54oC(highest) 127mm

Branch length (cm)

Leaf fresh weight (g)

Leaf dry weight (g)

Leaf width was measured by centimeter ruler.

Leaf length was measured by centimeter ruler.

Chlorophyll and Carotenoids estimation

Ascorbic acid estimation

Fresh wt. of sample× 1000

Amino acid content estimation

Totle free aminoacid= reading× vol. of extract× dilution factor

Wt. of sample× 1000

Total elemental %= C*(mg)× soln. vol(ml)

10× aliquot (ml) × sample wt.(g)

C*=mg element obtained from caliberation curve

Leaves fresh weight

leaves fresh wt. (g)

Sites Mean S.D S.E

Multan 8.905 ± 0.43562 ± 0.21781

Muzafergerh 5.815 ± 0.42273 ± 0.21136

Sites 91.587 3 30.529 39.9740*** 3.862548

Residual 6.8735 9 0.76372

*** = highly significant

Sites Mean S.D S.E

Multan 2.3175 ± 0.22618 ± 0.11309

Muzafergerh 2.235 ± 0.18930 ± 0.09465

Sites 4.67353 3 1.55784 3.02498 NS 3.86255

residual 4.63493 9 0.51499

Sites Mean S.D S.E

Multan 50.375 ± 2.926175 ± 1.463087

Muzafergerh 43.875 ± 3.275541 ± 1.63777

Sites 5428.543 3 1809.514 232.1461*** 3.862548

Residual 70.1525 9 7.794722

NS= Non significant *** = highly significant

leaf width (cm) 39

Sites Mean S.D S.E

Multan 40.6 ± 3.22594 ± 1.61297

Muzafergerh 39.075 ± 2.42676 ± 1.21338

Sites 4059.832 3 1353.277 249.9351*** 3.862548

Residual 48.73063 9 5.414514

*** = highly significant

Sites Mean S.D S.E

Multan 0.50471 ± 0.14297 ± 0.07148

Muzafergerh 0.35087 ± 0.10928 ± 0.07148

Table3.5b: Analysis of variance of chlorophyll a (mg/g) in leaves of Moringa oleifera under

Sites 12.35829 3 4.119429 3.862548

NS= non significant

***= highly significant

Sites Mean S.D S.E