Journalof Horticultureand Plant Research Vol 32018

Journal of Horticulture
and Plant Research
Volume 3
2018
Journal of Horticulture and Plant Research
ISSN 2624-814X
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Table of Contents
Arbuscular Mycorrhizal Fungi (AMF) as Buffer for Heavy Metals Phytoextraction by

Cucurbita maxima Duch. Grown on Crude Oil Contaminated Soil
O.G. Okon, J.E. Okon and G.D.O. Eneh 1
Response of Mung Bean Crop to Different Levels of Applied Iron and Zinc
A. Jamal, M.I. Khan, M. Tariq and M. Fawad 13
In Vitro Regeneration of Medicinally Important Shrub Carissa Opaca from Shoot Apices
and Nodal Segments
A. Ahmad, B.H. Abbasi and M. Zia 23
Screening of Sunflower Genotypes for Potassium Use Efficiency in Irrigated Soil Condition
S.B. Jamro, N.A. Talpur, M.K. Sootahar, Z.U.H. Shah, M.K. Sootahar and A.A. Panhwar 30
Sex Determination in Nutmeg Seedlings Using Scar Primers
F.D. Mintah 40
Journal of Horticulture and Plant Research Submitted: 2018-02-27
ISSN: 2624-814X, Vol. 3, pp 1-12 Revised: 2018-06-16
doi:10.18052/www.scipress.com/JHPR.3.1 Accepted: 2018-07-12
© 2018 SciPress Ltd., Switzerland
Arbuscular Mycorrhizal Fungi (AMF) as Buffer for Heavy Metals

Phytoextraction by Cucurbita maxima Duch. Grown
on Crude Oil Contaminated Soil
O.G. Okon1,a*, J.E. Okon2,b, G.D.O. Eneh3
1
Department of Botany and Ecological Studies, University of Uyo, Uyo, Nigeria
2
Biological Sciences Department, Akwa Ibom State University, Ikot Akpaden, Nigeria
3
Department of Science Technology, Akwa Ibom State Polytechnic, Ikot Osurua, Nigeria
a
okonokongodwin@yahoo.com, bokjunior4zeeb@gmail.com
Keywords: Cadmium, Copper, Chromium, Crude oil, Cucurbita maxima, heavy metals, Lead,
Rhizophagus irregularis, Zinc.
Abstract. This study evaluated the influence of Arbuscular Mycorrhizal (Rhizophagus irregularis)
fungi inoculation (M) on the growth of Cucurbita maxima and as a buffer against phytoextraction of
selected heavy metals (HM) (Zn, Cu, Cr, Cd and Pb) from a soil polluted with crude oil (C). The
experiment was set up using four soil treatments, in triplicates C+ M-, C+ M+, C- M+ and C- M-
(control without crude oil and inoculum). The shoot length, petiole length, number of nodes, leaf
area and percentage germination of C. maxima were significantly (p=0.05) reduced in uninoculated
crude oil treatment (C+ M-), unpolluted mycorrhizal inoculated treatments (C- M+) showed
remarkable response in growth parameters above the control (C- M-), while the polluted and
inoculated treatment (C+ M+) showed significant (p=0.05) increase in growth parameters when
compared to the polluted uninoculated treatment (C+ M-). Heavy metals analysis revealed a
significant (p=0.05) difference in the heavy metal accumulation of C. maxima. The heavy metals
analyzed in this study are present thus in C. maxima; Zn>Cu>Cr>Pb>Cd. Crude oil polluted
uninoculated treatment (C+ M-) recorded the highest concentrations of heavy metals than crude oil
polluted inoculated (R. irregularis) treatment (C+ M+). Mycorrhizal inoculated unpolluted
treatment (C- M+) and unpolluted uninoculated treatment (C- M-) indicated the lowest heavy metal
concentrations. Inoculation with R. irregularis significantly (p=0.05) reduced heavy metals uptake
by C. maxima as observed in this study. Also, the downbeat effect of crude oil on AM Fungi root
colonization of C. maxima by R. irregularis was observed in polluted and inoculated treatment. HM
often accumulate in the top soil, therefore, are available for uptake by plants via roots, which is a
major entry point of HM that ultimately affects different physiological processes. AM fungi can
impinge on the chemical properties of heavy metals in the soil, their absorption by the host plant,
and their allocation to different plant parts, affecting plant growth and the bioremediation process,
thus making the AM fungi a suitable buffer for mitigating heavy metal stress on C. maxima.
Introduction
Plants and environments are faced with diverse and concurrent types of stresses namely;
heavy metals, water logging, heat, salinity, drought, etc. adversely affecting plant growth and the
environment. The incidence of different microbes in the soil can be used as a great tool to augment
the efficiency of plants and the environment through symbiotic association. Diverse soil
microorganisms including arbuscular mycorrhizal (AM) fungi, plant growth-promoting
rhizobacteria (PGPR), and endophytic bacteria can positively enhance plant growth under diverse
circumstances including stress as well as the properties of the environment [1, 2]. Anthropogenic
actions severely affect our natural habitats. The re-establishment of such stress degraded habitats
via sustainable, low input cropping systems with the aim of maximizing yield of crop plants is
required for crop productivity. Thus, the integration of the innate roles of these advantageous
microbes in sustaining soil productiveness and plant productivity is gaining much more awareness
[3].
2 Volume 3
Heavy metals stress is among the most important stresses decreasing the efficiency of plant
and the environment [2]. Heavy metals (53 elements) are determined according to their density
(>5 g/cm3) [4]. Some of the heavy metals together with iron (Fe), zinc (Zn), manganese (Mn),
copper (Cu), and nickel (Ni) are essential for plant growth and yield production. Among the main
vital functions of such heavy metals in plant are (a) the catabolization of enzymatic and redox
activities, (b) the transfer of electron, and (c) as the main part of DNA and RNA metabolism [5].
The other heavy metals like lead (Pb), nickel (Ni), arsenic (As), etc., which are also the sources of
contamination, do not have any functions in living organisms. The elevated concentrations of these
heavy metals can adversely impinge on the metabolism of plant and soil microbes and hence the
food chain and the ecosystem [6]. The sources of heavy metals in the soil are industrialization and
urbanization as well as the unsuitable disposal of wastes, which has increased the concentration of
heavy metals within the environment [2]. Among such activities the anthropogenic activities
including fertilization, the use of herbicides, wastes, and sewage sludge are also among the most
important sources of environmental contamination. However, mining is the most important source
of trace element in the environment [2, 7].
Arbuscular mycorrhizal fungi (AMF), are beneficial root symbionts belonging to the phylum
Glomeromycota [8], they are ubiquitous in the environment with little host specificity and extensive
geographic allotment which are associated with majority of terrestrial plant species [9]. AMF plays
an essential role in the ecosystems steadiness by enhancing the adjustment, growth, and nutrition of
plants under unpleasant environmental settings [10, 11]. Different mechanisms are used by
mycorrhizal fungi to detoxify the adverse effects of heavy metals on the environment and on the
growth of the host plant including (a) the production of chelating products, (b) the interaction with
the plasma membrane, and (c) the effects of the cell wall components of the fungi and the plant.
Such mechanisms can be conducted by the following [2]: (a) Plant growth and the subsequent
dilution of heavy metals in the tissues, (b) The production of organic products such as organic acids
by plant roots, which prevents the uptake of heavy metals from the rhizosphere by chelating,
precipitating, and binding, (c) The selective activity of plasma membrane in the absorption or
desorption of heavy metals, (d) The retention and immobilization of heavy metals by the fungal
hyphae and plant roots, (e) Chelating heavy metals by metallothioneins in the cytosol of both fungi
and the host plant, (f) The activity of specific or non-specific carriers and plasma membrane pores
(both fungi and host plant) (g) Heavy metal sequestration by plant and fungi cellular vacuoles, (h)
Transfer of heavy metals by the fungal hyphae, (i) The exchange of heavy metals amongst the fungi
and the host plant and (j) Active transfer of heavy metals by the specific and non-specific pathways
in both fungi and the host plant [12, 13].
Cucurbita maxima belongs to the family Cucurbitaceae, they are prostrate or climbing,
annuals, they usually reach a length of about 4 meters or further. They possess hispid leaves, which
are rounded, about 15 to 30 cm in diameter, their base has a heart-shape [14]. In many countries in
tropical Africa and worldwide, there have been reports of the distribution of C. maxima [15]. It
northern Nigeria, C. maxima is cultivated mainly for its fruits, the leaves and fruits are the main aim
of C. maxima cultivation in southern Nigeria [16]. While the other two species C. pepo and C.
moschata are integral parts of diets of indigenous people of this region [17]. This research was
carried out to explore the potential use of arbuscular mycorrhizal fungi (AMF) as a buffer to
mitigate phytoextraction of heavy metals by C. maxima from soils contaminated with crude oil.
Materials and Methods

Study Area
This research was carried out in Uyo Local Government Area of Akwa Ibom State, Nigeria.
Uyo is a city in South-South Nigeria found between latitude 5.02°N and longitude 7.92°E; it has an
average temperature of 25.1-27.8°C, an annual rainfall of 3300 mm. The plant (Cucurbita maxima)
used for this research work was identified by a plant taxonomist in the Department of Botany and
Ecological Studies, University of Uyo, Uyo, Nigeria.
Journal of Horticulture and Plant Research Vol. 3 3
Sources of Crude Oil and Experimental Soil

The crude oil used for this research was gotten from the Nigerian National Petroleum
Commission (NNPC), Aba Road, Port Harcourt-Nigeria. The experimental soil was excavated from
the Department of Botany and Ecological Studies Botanic Garden, University of Uyo-Nigeria.
Source of Arbuscular Mycorrhizal (AM) Fungi
AM Fungi Rhizophagus irregularis (60–65 spores per 5 g) was purchased from International
Institute of Tropical Agriculture (IITA) Ibadan-Nigeria.
Pollution and Amendment Treatments
10 kg of the sandy-loam were weighed using a weighing balance (Ohaus Triple Beam 750-SO
Weighing Balance). Polluted soils were obtained by mixing thoroughly 10 kg of sandy-loam with
50 ml of crude oil and left undisturbed for one week, while unpolluted garden soil was used as
control.
Propagation and Mycorrhizal Inoculation
Arbuscular Mycorrhizal fungi Rhizophagus irregularis was propagated on Zea mays planted
and grown in a greenhouse for about 16 weeks. The colonized maize roots were used as an
inoculum (25 g fresh weight per pot containing approximately 130 spores) was placed in the pot at
15 cm depth, before planting.
Sterilization of Seeds for Planting
Seeds of C. maxima were surface sterilized with 0.01% mercuric chloride solution for 30
seconds, meticulously washed severally with clean distilled water [18]. Five (5) seeds of C. maxima
were sown directly in each perforated buckets containing polluted and unpolluted garden soil. On
seedling emergence, the plants were thinned to 3 per pot.
Experimental Setup
This experiment was set up using a complete block design (CBD) having three (3) replicates
for C. maxima.
Treatments Meaning
C- M- - Crude Oil, - Mycorrhiza
C+ M- + Crude Oil, - Mycorrhiza
C+ M+ + Crude Oil, + Mycorrhiza (Rhizophagus irregularis)
C- M+ - Crude Oil, + Mycorrhiza (Rhizophagus irregularis)
C- (- crude oil), C+ (+ crude oil), M- (- mycorrhiza) M+ (+ mycorrhiza).
Determination of Growth Parameters
Percentage seedling emergence was calculated as the seeds emerged from the soil five (5)
days after sowing. The % germination for each treatment was calculated using the formula:
Number of Emerged Seeds 100
n = % seedling emergence = x
Number of Seeds Sown 1
Measurement of growth parameters such as shoot length, root length, petiole length and internode
length were taken nine (12) weeks using a measuring tape (cm). Leaf number was counted. Leaf
area (LA) was determined using a graph by multiplying the leaf length by leaf width (widest
portion) with the correlation co-efficient (r) which was 0.72.
Heavy Metal Analysis
The powdered samples were digested with very strong acids, perchloric acid (HCLO4) and
nitric acid (HNO3) and this is called Wet digestion method. The digested samples would be read
using Unicam 939 Atomic Absorption Spectrophotometer.
4 Volume 3
Wet digestion: 1 g of the powdered sample was weighed into a digestion flask, 20 ml of nitric
acid was added and 10 ml perchloric acid was also added, a drop of concentrated Sulphuric acid
added also. The mixture was allowed to stand for 30 minutes and then digested using digestion
chamber in the laboratory until the colour turned white. This showed that the samples were
digested. They were allowed to cool. Thirty (30) ml of distilled water were added and were filtered.
The filtrate was made up to 50 ml solution with distilled water. The digested solution was stored in
a sample bottle ready for the heavy metals analysis using atomic absorption spectrophotometer. The
concentrations of Chromium (Cr), Copper (Cu), Lead (Pb), Cadmium (Cd) and Zinc (Zn) in the
powdered samples were determined according to the recommended methods described by the
Association of Official Analytical Chemists [19].
Assessment of Arbuscular Mycorrhizal Colonization
Feeder roots of about 2–4 cm of Cucurbita maxima were collected and fixed in 50% ethanol
and stored for colonization assessment. The fixed roots were rinsed in tap water before clearing in
10% KOH w/v and autoclaved for about 15 minutes at 121°C. Cleared roots were collected on a
sieve and severally rinsed with tap water before staining. Staining of the plants roots was carried out
using a very recently improvised method of staining using ink and vinegar. This staining solution is
made up of a 5% ink watered down in vinegar (5% acetic acid). Staining with blue writing ink was
done by soaking the roots segments in the ink and allowing the stained roots in the staining solution
at normal room temperature for a day. The stained plant roots become more visible after destaining
soaked roots in 50% glycerol for about 1 hour [20].
After the stained roots had been cleared of stains, root length was measurement was taken
concurrently with mycorrhizal colonization by a gridline intersection procedure independently by
viewing slides with a dissecting microscope; where the stained roots are randomly dispersed in a
Petri dish plate of about 9 cm diameter with grid lines. Quantifying intersections was done by
scanning alongside these grid lines using a dissecting microscope linking grid lines and roots which
are selected as either colonized or non-mycorrhizal. The fraction of the root length that is
mycorrhizal and the total root length can then be calculated from a conversion factor resulting from
the total length of grid lines and the area of the dish. A minimum of 100 intersections was used to
assess the stained root samples; the stained root samples were re-ordered randomly and counted
numerous times. Percentage mycorrhizal colonization was calculated using standard methods
described by Giovannetti and Mosse [21].
Total number of roots infected intersecting gridlines
MC = × 100
Total number of roots intersecting gridlines
Statistical Analysis
Data obtained for various growth parameters subjected to pollution and AMF inoculation
treatments were analyzed using the Statistical Package for Social Sciences (SPSS) version 20.0 and
means alienated with standard error of means. Analysis of variance (ANOVA) was also carried out
on data collected for different heavy metal contents of this research. GraphPad Prism analytical
software package, version 6 was employed for the reason mentioned above. Duncan’s Multiple
Range Test was used to separate means at p=0.05.
Results and Discussion

Duncan Multiple Range comparisons of the means for every treatment showed that there was
significant (p=0.05) difference among treatments analyzed (C- M-, C+ M-, C+ M+ and C- M+)
(Fig. 1, Fig. 2 and Fig. 3). This indicated a significant difference in the growth parameters
(percentage germination, shoot length, petiole length, leaf number, internode length and leaf area of
the C. maxima seedlings used in this study (Fig. 1).
120 80
(A ) (B ) a
a
s h o o t le n g t h ( c m )
110
60 b
% g e r m in a t io n
a b
100
90
b 40
80
c 20
c
70
60 0
- - - -
M + + M + +
M M M M M M
- + - - + -
C C + C C C + C
C C
T re a tm e n ts T re a tm e n ts
25 30
(C ) (D ) a
p e t io le le n g t h ( c m )
20 a
le a f n u m b e r
b 20 b b
15 b
10 c
10
c
5
0 0
- - +
+
-
M
-
+
+
M
M M
M
- +
M
M
+ -
+
C C
-
-
+
C
C
C
C
25 150 (F )
(E )
a
in t e r n o d e le n g t h ( c m )
20
le a f a r e a (c m )
2
a
100 b
b b
15
b
10
c 50
5
c
0 0
-
-
+
-
+
+
+
M
M
M
M
M
M
M
M
+
+
-
-
-
-
+
C
+
C
C
C
C
C
C
C
Figure 1. Effect of crude oil pollution and mycorrhizal (R. irregularis) inoculation on (A)
percentage germination (B) shoot length (C) petiole length (D)
leaf number (E) internode length and (F) leaf area of C. maxima
The shoot length, petiole length, number of nodes, leaf area and percentage germination of C.
maxima were drastically (p=0.05) reduced in uninoculated crude oil polluted treatment (C+ M-),
unpolluted mycorrhizal inoculated treatments (C- M+) showed remarkable response in growth
parameters above the control (C- M-), while the polluted and inoculated treatment (C+ M+) showed
noteworthy (p=0.05) increase in growth parameters when compared to the polluted uninoculated
treatment (C+ M-). The results agree with the work of Ekpo et al. [22] who reported reduction in
6 Volume 3
shoot length and general growth of soybean (Glycine max) as a result of unsatisfactory soil
conditions associated with reduction in pore spaces thus increasing the demand of oxygen by
hydrocarbonoclastic microorganisms. Olusola and Anslem [23] also observed reduction in general
growth parameters in Amaranthus hybridus cultivated on crude oil polluted soil. Results of this
research revealed that inoculation of C. maxima with R. irregularis showed more prolific growth
than uninoculated treatments despite soil pollution by crude oil. Similar researches are in line with
the observations of this research eg, Zea mays [24-26], Lycopesicum esculentus [27] and
Amaranthus hybridus [23]. These results clearly show that crude oil contamination can hinder the
growth and metabolic activities of plants, however, inoculation with AM fungi accelerates the
biodegradation of several organic chemicals like; polycyclic aromatic hydrocarbons (PAHs) which
are constituents of crude oil, thereby releasing functional mineral nutrients resulting to an improved
in the growth and metabolism of plants [3].
Heavy metals analysis reveals a significant (p=0.05) difference in the heavy metal
accumulation of the C. maxima (Fig. 2). The heavy metals analyzed in this study are present thus in
C. maxima; Zn>Cu>Cr>Pb>Cd (Fig. 2). Crude oil polluted uninoculated treatment (C+ M-)
recorded the highest concentrations of heavy metals than the oil polluted inoculated (R. irregularis)
treatment (C+ M+). Mycorrhizal inoculated unpolluted treatment (C- M+) and unpolluted
uninoculated treatment (C- M-) indicated the lowest heavy metal concentration (Fig. 2). This is in
line with the work of Okon [28] who reported huge amount of heavy metals accumulation in
Cucurbita maxima such as Zinc, cadmium, copper and chromium with increased crude oil pollution
levels, with zinc showing the highest concentration. This observation agrees with the findings of
Agbogidi et al. [29] who reported the accumulation of iron, zinc, cadmium, manganese, lead and
cadmium contents in maize (Zea mays L.) cultivated in soil polluted with crude oil which led to
poor growth and yield reduction. Lead and cadmium prevents mineral uptake by either synergistic
or antagonistic reactions leading to poor growth [30]. Similarly, Ogbuehi et al. [31] reported the
accumulation of zinc, lead and nickel on seeds and leaves of groundnut (Arachis hypogea)
cultivated on spent engine oil polluted soil. Also, Ogbuehi et al. [32] Ogbuehi et al. [33] and
Dushenkove et al. [34] reported separately that certain plants including cassava can extract heavy
metals from crude oil polluted soils. Ekundayo et al. [35] in their study on the effect of spillage of
crude oil on the growth, yield and nutrient assimilation of Zea mays, showed that soils contaminated
with crude oil delayed germination and general growth of plants. Chukwuemeka et al. [36] reported
the accumulation of Nickel, Vanadium, Cadmium, Lead, Manganese, Iron, Cobalt and Zinc in
Telfairia occidentalis. Osu et al. [37] corroborates this fact by stating that Vernonia amydalin,
Telfairia occidentalis and Amaranthus are good accumulators of heavy metals. Heavy metals that
are found in arable crops, have a latent danger to man via the dietary pathway [38].
250 a
(a ) 0 .8 a
(b )
200 b
c
c a d m iu m (m g /k g )
z in c ( m g /k g )
0 .6
150 d
0 .4 b
100
c
d
50 0 .2
0 0 .0
- - -
M + + - + +
M M M M M
- + - - + M M
C C + C C C + -
C C C
Figure 2. Effect of crude oil pollution and mycorrhizal (R. irregularis) inoculation on (a) zinc (b)
cadmium (c) chromium (d) copper (e) lead concentration of C. maxima
Inoculation with R. irregularis significantly (p=0.05) reduced heavy metals uptake by C.
maxima as observed by this study. The exclusion of Pb in tissues of C. maxima in this research is
corroborated by the work of Liasu et al. [39] who reported that AMF inoculation led to Pb exclusion
in Zea mays. Schneider et al. [40] also investigated the role of mycorrhizal fungi on plant diversity
and bioremediation in areas polluted with Pb observed a noticeable reduction in Pb uptake hence,
subsequently recommending the use of the fungi for bioremediation techniques such as
biostabilization. Similarly, Hristozkova et al. [41] studied the effects of mycorrhization on the
8 Volume 3
removal of Cd and Pb from a contaminated soil; the authors examined the effects of mycorrhizal
fungi on the oil composition of Origanum majorana, as well as the antioxidant activity and the
uptake and buildup of heavy metals by plants; they reported decreased uptake of Cd and Pb. Galli et
al. [42] reported reduction differences in Cu uptake among mycorrhizal and non-mycorrhizal Zea
mays. Abdul et al. [3] reported that AM fungi protect Zea mays from Cu-toxicity, Therefore,
corroborating the results of this study.
Abd-Allah et al. [43] reported that Cd stress adversely affected plant growth, the chlorophyll
content, and the stability of the cellular membrane. Similarly, Cabral et al. [44] reported that
mycorrhizal fungi inoculation resulted in fewer uptakes of Cd and Pb. However, the fungi were
capable to assuage the stress by positively affecting the parameters earlier mentioned. Gianinazzi et
al. [45] reported that Glomus sp. decreased Cd concentrations in clover shoots and also did have
impact on plant growth. Cd elimination particularly in the unpolluted treatments is possibly bridged
by AM inoculation via a network of fungal hyphae that adsorb the metal and keep it sequestered on
active sites of the hyphal wall [46]. Wu et al. [47] also investigated the effects of AM fungi on the
exclusion of Cr from a polluted soil resulting in the significant immobilization of Cr and its
subsequent reduction. Likewise, Kanwal et al. [48] studied the effects of mycorrhization on the
growth of wheat plants in contaminated soil, different plant parameters including plant growth, root
colonization, the content of heavy metals; Mycorrhization significantly decreased the concentration
of Zn in plant aerial as also observed in this study.
The efficiency of AM fungi in the removal of heavy metals from the polluted soils and plant
aerial part is different, and some mycorrhizal fungi may be more efficient compared with the other
types of fungal species. The allocation of the heavy metals by AM fungi to different parts of the
host plant is also different as some heavy metals may concentrate in the roots and some may
concentrate in plant aerial part [49, 50]. The two most important mechanisms by which the
mycorrhizal fungi are able to reduce the contamination of polluted areas are phytostabilization and
phytoextraction. During the uptake of heavy metals by mycorrhizal fungal hyphae, a couple of
physiological mechanisms including (1) the expression of proteins such as metallothioneins and
glomalin, (2) the retention of heavy metals by fungal spores, (3) chelation/complexation of heavy
metals in the cellular membrane, and (4) the molecular expression of the genes results in the
activation of the phytostabilization process [51].
Heavy metals (HM) frequently build up in the top soil, therefore, are available for uptake by
plants via roots, which is a major entry point of HM that ultimately affects different physiological
processes [52]. Cd is very toxic even at very low concentrations and do not have any known
bioimportance in the biochemistry and physiology of humans [53], and long term exposure results
in fragile bones and decreased bone strength [54], renal dysfunction, characterized by tubular
proteinuria [55] in humans, severe irritation of the stomach leading to vomiting and diarrhea which
can sometime result in death, kidney damage [54]. Cadmium is also a proven human carcinogen
[54]. Thus, households or individuals that have been making uses of vegetables from the crude oil
polluted sites may have predisposed themselves to serious risk of cadmium uptake, bioaccumulation
and biomagnification.
The negative effect of crude oil on AMF root colonization of C. maxima by R. irregularis was
observed in polluted and inoculated treatment (Fig. 3). Prior studies indicates that environmental
stressors such as; temperature, soil moisture, pH, tilling, root exudates and inorganic/organic
contaminants (eg. Petroleum hydrocarbons)[57]. In this regard, diverse researchers have
investigated extensively and reports that petroleum hydrocarbons have a detrimental effects on
AMF fungal structures [57]; in Cichorium intybus there was severe decrease in percentage hyphae
with increase of concentrations of 35, 70, 140, and 280 μM of benzo(a)pyrene [58]; in Allium
porrum there was drastically lower hyphae number, and arbuscule structure of Gigaspora rosea was
observed in the at 200 μl of ethylbenzene [59]. The reduction in root colonization of C. maxima by
R. irregularis observed in this study could be as a result of drastic reduction of moisture and oxygen
amidst aggregates of soils and roots, it could also be aligned [60] to the fact that the root cortical
cells under any environmental stress contains petite amounts of sugar on which AMF depends on to
carry out its metabolic processes. The minute quantity of photosynthates is a drawback in terms of
the plant ability to adapt; additionally, 30% of the carbon fixed (sugars) during photosynthesis is
used by AMF [61].
Figure 3. Mycorrhizal Colonization of C. maxima in Soil Contaminated with Crude Oil
Conclusion
Inoculation of Cucurbita maxima with AM fungus (Rhizophagus irregularis) enhanced the
vegetative growth parameters both in crude oil polluted and unpolluted (control) soils. AMF
inoculation inhibited the phytoextraction of heavy metals (Zn, Cu, Cr, Pb and Cd) from
contaminated soils. Heavy metal stress, which is the result of industrialization and excess use of
chemicals, is a major concern affecting large areas of the world. Although different methods have
been used to mitigate the contaminated environments with heavy metals, the use of biological
methods, which is the single or the combined use of plants and microbes, has been among the most
effective ones. Arbuscular mycorrhizal fungi have some terrific abilities, which make them endure
the stress of heavy metals and assuage the unfavorable effects of the stress on plant growth and on
the environment. AM fungi can impinge on the chemical properties of heavy metals in the soil, their
absorption by the host plant, and their allotment to different plant parts, affecting plant growth and
the bioremediation process, thus making the AM fungi a suitable buffer for mitigating heavy metal
stress on plants.
Conflict of Interest
The authors declare that there is no conflict of interest between them.
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Response of Mung Bean Crop to Different Levels

of Applied Iron and Zinc
Aftab Jamal1,*, Muhammad Ismail Khan1,
Muhammad Tariq1 and Muhammad Fawad2
1
Department of Soil and Environmental Sciences, University of Agriculture, Peshawar, Pakistan
2
Department of Weed Science, University of Agriculture, Peshawar, Pakistan
aftabses98@gmail.com
Keywords: Mung bean, Nodules, Fe, Zn.
Abstract. Fertilization of Mung bean (Vigna radiata L.) is one of the most crucial management
technique which effects crop growth and yield. Therefore the present study was carried out at
Agricultural Research Station Kohat under rain fed conditions during spring 2017, to assess the
response of mung bean (Vigna radiate L.) to three levels of iron (0, 2 and 5 kg ha-1) and three levels
of zinc (0, 5 and 10 kg ha-1). The experiment was laid out in randomized complete design with split
plot arrangement and replicated three times. The results revealed that application of Fe at the rate
5 kg ha-1 and Zn at the rate 10 kg ha-1 significantly increased biological yield, grain yield, straw
yield, nodule numbers and weight by 5624 kg ha-1, 968 kg ha-1, 4655 kg ha-1, 35 and 0.67g
respectively whereas the interaction was found non-significant. The nitrogen content in grains and
straw was also significantly increased by 2.22% and 3.56% respectively with application of Fe at
5 kg ha-1 and Zn at 10 kg ha-1, however their interaction was also found non-significant. Similarly,
the plant nitrogen uptake was also significantly increased by 323.33 kg ha-1 with application of Fe
at 5 kg ha-1 and Zn at 10 kg ha-1. It was concluded that Fe and Zn enhanced mung bean productivity.
Introduction
Mung bean (Vigna radiata L.) belongs to family Fabaceae and a well known crop of Pakistan
enriched in proteins, fibers and in vitamin A [1], Pakistan produced about 93 thousands t/ha of
mung bean [2]. In Pakistan, mung bean is grown during July to October and March to June seasons.
Although it is grown in different crop rotations, about 75% cultivation follows mung bean - wheat
crop rotation. Punjab is the major mung bean growing province that alone accounted for 88% area
and 85% of the total mung bean production. [3], however the distinctive feature of mung bean is the
root nodules containing nitrogen fixing bacteria Rizobium which in turn improve soil fertility [4],
and required lesser irrigation as compared with other field crops [5]. On average annually mung
bean can fix about 300 kg ha-1 atmospheric nitrogen [6]. Despite of its importance mung bean got
little attention in Pakistan and yet there is a lot of work to do in improving the quality and quantity
of mung bean crop.
A balance fertilization of both macro and micro nutrients are equally important for achieving
profitable yield of crops [7]. Iron is an essential nutrient for crop growth involved in chlorophyll
synthesis and chloroplast development [8]. Although iron content both in soil and plants are high
than S content and even P, yet its availability is low for plants and hence Fe deficiency is a common
problem [9]. So far the role of iron in nodulation and symbiotic nitrogen fixation is not clear,
however many investigator reported poor nodulation in absence of Fe [10, 11], but these reports
failed to explain the initiation of nodules and development. Iron is a key part of several key proteins
like nitrogenase, leghaemoglobin and ferredoxin [12]. Deficiency of iron reduced the initiation as
well as development of nodules in legumes [13]. Therefore, the deficiency of iron in legumes is a
clear sign of nitrogen deficiency too.
Zinc is a basic micro nutrient and performs several key roles in plant nutrition as well as in
human nutrition and its deficiency has an adverse effect on reproductive system [14]. Zn has a
dominant role in metabolism of plant hormone called auxin hence promote plant nutrition [15, 16].
Mung beans are very sensitive to Zn deficiency in alkaline soil like Pakistan [17]. Many
investigators reported Zn deficiency in Pakistani soils [18-20].
14 Volume 3
The importance of Zn for nodulation and yield of mung bean was also reported by various
researchers [21-24]. Similarly, [25] also reported more weight of nodules with application of Zn.
Maximum nodulation was reported in lentil at application of 9 kg ha-1Zn [26].
Therefore, keeping in view, the nutritional importance of iron and zinc for mung bean crop, a
field experiment was carried out at Kohat Research Station, to evaluate the effect of different levels
of Zn and Fe on mung bean crop.

The experiment was carried out at Agricultural Research Station Kohat under rain fed
conditions during spring 2017, to study the effect of iron and zinc on the yield, nodulation and
nitrogen uptake by mung bean (Vigna radiate L). Mung bean cultivar KM-1 was used as a test crop.
The mung bean seeds were sown in randomized complete design with split plot arrangement and
replicated three times. Size of the plot was 3 x 5 m2. Each plot was comprised of 10 rows containing
row to row distance of 30 cm and plant to plant distance was of 20 cm. Different levels of iron at
the rate of 0, 2 and 5 kg ha-1 and zinc at the rate of 0, 5 and 10 kg ha-1 were applied in the form of
iron sulfate and zinc sulfate, respectively. A basal dose of 25 N, 60 P2O5 and 60K2O kg ha-1 was
also applied in the form of urea, di-ammonium phosphate and potassium sulfate before sowing.
These fertilizer doses were calculated according to [2, 26, 32 and 33] with some little modification.
Before fertilizer application a composite soil sample was collected for the determination of physio-
chemical characteristics and desired nutrients status for the test soil. All the agronomic and culture
practices were performed. Data on nodules number and nodules weight was recorded at appropriate
time (between maximum plant biomass and flowering stage). Five plants were selected randomly
from each treatment and carefully uprooted along with soil to avoid damage the roots. Then it was
placed in plastic bucket containing water and washed carefully the roots. After washing nodule
number was counted on each plant roots. After those nodules were removed from plant roots and
weighed by an electronic analytical balance (A balance weighs up to 200 g i.e. 4 decimal places in
g, and reads to 0.1 mg). At maturity stage the plants were harvested to determined biological yield
and grain yield. The total-N concentration in straw and grain was determined by Kjeldhal apparatus
as described by [27]. The Nitrogen uptake in grain or straw was determined by multiplying their
percent concentrations with the corresponding yield. The total nitrogen uptake was obtained by
adding up their respective uptake in grain and straw and was expressed in kilogram per hectare. All
the data obtained were subjected to Statistix 8.1 software. After that LSD test of significance was
used to compare the treatment differences at P<0.05 level of probability.
Results and Discussions

The physio-chemical attributes of the soil revealed that soil was sandy loam in texture,
alkaline in reaction; low in organic matter, calcareous in nature, the zinc and iron content were
found low to optimum level (Table 1).
Table 1. Physico-chemical characteristics of soil before mung bean sowing
Physico-chemical properties Units Values
Sand % 55
Silt % 25
Clay % 20
Textural Class Sandy loam
Organic matter % 0.72
pH - 7.75
Lime % 6.2
EC m mhos 260
Total nitrogen % 0.04
Iron ug g-1 1.51
Zinc ug g-1 0.59
Nodules number
Number of nodules as influenced by different levels of Fe and Zn are presented in table 2.
Both Fe and Zn applied levels significantly increased the no of nodules. Based on result the
maximum no of nodules plant-1 of 32 were noticed at application of Fe at 5 kg ha-1, similarly Zn
application also improved the no of nodules plant-1 linearly from 24 at 0 kg ha-1 to 30 at 10 kg ha-1.
The interaction effect of Fe and Zn was not significantly affecting the number of nodules in mung
bean plants. The maximum, 35 nodules plant-1 was observed at plot receiving Fe and Zn 5 kg ha-1
and 10 kg ha-1 respectively and 20 nodules plant-1 at control. Our results was in lined with the
published literature it has an established criterion that Fe has dominant effect on nodule formation
which ultimately increased the no of nodules plant-1. [13] Reported that iron deficiency reduced
initiation and development of nodules, similarly [28] also reported positive effect of iron and zinc
on number of nodules. Poor nodulation in Lupinus albus L was also reported by [29] in Fe
deficient soil. Application of micronutrients (Zn, Fe & Mo) along with Rhizobium improved
nodulation and yield of mung bean [2]. As nodules are the residing place for nitrogen fixing
bacteria so it enhances nitrogen fixation in legumes crops.
Table 2. Number of Nodules per plantof mung bean as affected by different levels of Iron and Zinc
Zn Levels ---------Fe Levels (kg ha-1)---------
(kg ha-1) 0 2 5 Mean
0 20 23 29 24 c
5 23 27 33 28 b
10 25 30 35 30 a
Mean 23 c 27 b 32 a
LSD(0.05) of Nodules plant-1 for Fe= 394.98
LSD(0.05) of Nodules plant-1for Zn= 655.19
LSD(0.05) of Nodules plant-1 for Fe*Zn= 1134.8
Weight of nodules (gm)
The weight of nodules increased simultaneously with the application of both Fe and Zn levels
(Table3). Maximum weight of 0.61 gm was recorded at 5 kg ha-1 Fe application and 0.57gm at
10 kg ha-1 Zn application. As in table 2, it was cleared that Fe application speed up the nodule
formation which on other hand also increased its weight which is not strange. Our results was also
supported by many researchers [30] also resulted increased in weight of nodules with the
application of Zn. More nodules formation is associated with micro nutrients application [2].It is an
established criterion that Fe deficient crop appeared nitrogen deficiency. The interaction of Fe and
Zn levels was also significantly increases the weight of nodules. The maximum weight of 0.67 gm
was at full dose of Fe and Zn 0.31 gm was observed in control (Table3). Our results were in lined
with published literature, [28] stated that Zn application significantly increased the nodules number
and ultimately weight. [10] Reported that Fe application significantly increased the number and
weight of nodules. [31] Conformed that Zn application enhance nodulation which ultimately
improve nodules biomass.
Table 3. Weight of Nodules (in grams) of mung bean as affected by different levels of Iron and Zinc
0 20 23 29 24 c
5 23 27 33 28 b
10 25 30 35 30 a
Mean 23 c 27 b 32 a
LSD(0.05) of weight of Nodules for Fe= 0.0235
LSD(0.05) of weight of Nodules for Zn= 0.0311
LSD(0.05) of weight of Nodules for Fe*Zn= 0.053
16 Volume 3
Biological yield
Results revealed that both the Fe and Zn levels significantly increased the biological yield of
mung bean crop over control (Table 4). Iron applied at the rate of 5 kg ha-1 attributed the maximum
biological yield of 4533 kg ha-1 whereas the minimum of 3541 kg ha-1 was observed in control.
When averaged across the Fe levels, maximum biological yield of 4997 kg ha-1 was observed in the
plots treated with 10 kg Zn ha-1 while the minimum biological yield of 3192 kg ha-1 was noted in
untreated plots. The interactive effect of Fe and Zn was observed non-significant. The maximum
biological yield of 5624 kg ha-1 was noted in the plots receiving 5 and 10 kg ha-1 Fe and Zn
respectively whereas the lowest yield of 2823 kg ha-1 was observed in control (Table 4). Many
researchers reported significant increase in yield with the application of different levels of Fe. [11,
32] also reported an increase in biomass with the application of micro nutrients (Zn, B, Mo, Fe)
different levels. [33] Concluded the same results in mung bean. [34] Stated that micronutrients play
important role in growth and yield of legumes. [35] Reported that applied levels of (Fe + Zn)
combine and zinc alone significantly increased the crop yield.
Table 4. Biological yield (kg ha-1) of mung bean as affected by different levels of Iron and Zinc
0 2823 3010 3743 3192 c
5 3634 3944 4233 3937 b
10 4167 5200 5624 4997 a
Mean 3541 c 4051 b 4533 a
LSD(0.05) of BY for Fe= 406.47
LSD(0.05) of BY for Zn= 646.13
LSD(0.05) of BY for Fe*Zn= 1119.1
Grain yield
Grain yield was significantly increased with increasing levels of Fe and Zn (Table 5). On
average maximum grain yield of 636 kg ha-1 was obtained from the plots treated with high dose
5 kg ha-1 of Fe which was significantly higher than control. Results regarding Zn levels showed a
significant increased in grain yield with each increment. On average, maximum grain yield of
743 kg ha-1 was observed in the plots treated with 10 kg ha-1 Zn. The interactive effect of Fe and Zn
was also significant. On average, the maximum grain yield of 968 kg ha-1 was obtained for the
treatment receiving Fe at 5 kg ha-1 and Zn 10 kg ha-1(Table5 and Fig.1). Many published literatures
confirmed the results. [36] Reported an increase of 28.1% of mung bean grain yield with Zn
fertilization as compared with control. Zn application improves the growth and quality parameters
in mung bean [22, 37]. Increased in grain yield of mung bean with application of Zn and Mg was
also reported by [38]. Foliar application of iron significantly increased the grain yield of mung bean
[39]. Iron and zinc application increased the grain yield either applied to soil or applied as foliar
spray [40, 41].
Table 5. Grain yield as (kg ha-1) of mung bean as affected by different levels of Iron and Zinc
0 303 e 328 e 378 e 336 c
5 407 de 528 cd 562 c 499 b
10 517 cd 745 b 968 a 743 a
Mean 409 c 534 b 636 a
LSD(0.05) of GY for Fe= 98.152
LSD(0.05) of GY for Zn= 59.868
LSD(0.05) of GY for Fe*Zn= 103.69
1000
Grain Yield (kg ha-1) 800
600
0
400 5
10
200 10
5
0
0 0
2
5
Fe Level (kg ha-1)
Figure 1. Grain yield as (kg ha-1) of mung bean as affected by different levels of Iron and Zinc
Straw yield
On average, maximum straw yield of 3897 kg ha-1 was obtained from the plots treated with
high dose of 5 kg ha-1 of applied Fe that was statistically similar to the plot receiving Fe dose at
2 kg ha-1 while significantly higher than the control (Table 6). Zinc application at different levels
increased the straw yield of mung bean crop significantly. On average, maximum straw yield of
4253 kg ha-1 was observed in the plots treated with 10 kg ha-1 Zn and the minimum 2856 kg ha-1
was without Zn application. However, the interactive effect of Fe and Zn was found non-significant.
Maximum straw yield of 4655 kg ha-1 was recorded from treatment containing Fe at 5 kg ha-1 and
Zn at 10 kg ha-1 and that was statistically similar for the treatments receiving Fe at 2 kg ha-1 and Zn
10 kg ha-1 and the minimum yield of 2520 kg ha-1 was observed in control (Table 6). The result was
corroborated with published values; [42] reported that Fe levels significantly increased the straw
yield of cowpea. [33] Concluded that the seed and Stover yield of mung bean significantly
increased over control by the application of Fe. Micro nutrients application can increase the straw
yield of mung bean [43].
Table 6. Straw yields (kg ha-1) of mung bean as affected by different levels of Iron and Zinc
0 2520 2682 3365 2856 b
5 3227 3415 3672 3438 b
10 3650 4455 4655 4253 a
Mean 3133 b 3517 ab 3897 a
LSD(0.05) of SY for Fe= 394.98
LSD(0.05) of SY for Zn= 655.19
LSD(0.05) of SY for Fe*Zn= 1134.8
Grain Nitrogen (%)
Both Iron and Zn application significantly increased the nitrogen content in mung bean grain
(Table 7). Iron levels significantly affect the nitrogen content. Maximum nitrogen of 3.35 % was
recorded at 5 kg ha-1 Fe application and minimum of 2.95 % was at control. Zn levels showed
a significant increase in grain nitrogen with each increment. On average, maximum nitrogen content
of 3.35 % was observed in the plots treated with 10 kg ha-1 and minimum of 2.92 % was at without
Zn application. The interaction of applied Fe and Zn was found non-significant effect on grain
18 Volume 3
nitrogen content of mung bean. The maximum nitrogen of 3.56 % was recorded at 5 and 10 kg ha-1
applied Fe and Zn respectively and minimum of 2.65 % was at control. Our results were in lined
with [22, 44, 45, 23, 46 and 47] who reported increased in grain nitrogen with application of Zn
fertilization. Similarly, [2] also confirmed that N content in mung bean increased with
micronutrients (Zn, Fe & Mo) application.
Table 7. Grain Nitrogen conc (%) of mung bean as affected by different levels of Iron and Zinc
0 2.65 2.97 3.13 2.92 b
5 2.99 3.17 3.36 3.17 ab
10 3.20 3.28 3.56 3.35 a
Mean 2.95 b 3.14 ab 3.35 a
LSD(0.05) of Gain N for Fe= 0.3532
LSD(0.05) of Gain N for Zn= 0.2664
LSD(0.05) of Gain N for Fe*Zn= 0.4614
Straw nitrogen concentration (%)
Straw Nitrogen content as affected by different levels of Fe and Zn are presented in table 13.
Both Iron and Zn application significantly increased the mung bean straw nitrogen. Iron levels
significantly affect the nitrogen content. Maximum % Nitrogen of 1.82 % was recorded 5 kg ha-1 Fe
application and minimum of 1.51 % was at control. Results regarding Zn levels showed a
significant increase in straw nitrogen with each increment. On average, maximum nitrogen content
of 1.89 % was observed in the plots treated with 10 kg ha-1 and minimum was at without Zn
application. The interaction of applied Fe and Zn was found non-significant. The maximum %
nitrogen of 2.22 % was recorded at 5 and 10 kg ha-1 applied Fe and Zn respectively and minimum
of 1.35 % was at control. Published literature revealed that iron application increased the N content
of plants. [2] Reported that N content in mung bean plant increased by micronutrients (Zn, Fe &
Mo) application. Zn application reduced the P content however it increased crop nitrogen content
[23].
Table 8. Straw Nitrogen (%) concentrations of mung bean as affected by different levels of Iron and Zinc
0 1.35 1.51 1.55 1.47 c
5 1.52 1.66 1.72 1.63 b
10 1.66 1.82 2.22 1.89 a
Mean 1.51 b 1.66 ab 1.82 a
LSD(0.05) of straw N for Fe= 1.7988
LSD(0.05) of straw N for Zn= 3.2957
LSD(0.05) of straw N for Fe*Zn= 5.7084
Total nitrogen (Straw+Grain) uptake
Both Iron and Zn application significantly increased the nitrogen uptake in mung bean crop.
Iron levels significantly affect the nitrogen uptake. Maximum nitrogen uptake of 238.06 kg ha-1 was
recorded 5 kg ha-1 Fe application and minimum of 160.34 kg ha-1 was at control (Table 9). Results
regarding Zn levels showed a significant increase in nitrogen uptake with each increment. On
average, maximum nitrogen content of 263.99 kg ha-1 was observed in the plots treated with 10 kg
ha-1 and minimum of 141.58 kg ha-1 was at 0 kg ha-1 applied Zn. The interaction effect of applied Fe
and Zn was found non-significant. The maximum nitrogen uptake of 323.33 kg ha-1 was recorded at
5 and 10 kg ha-1 applied Fe and Zn respectively and minimum of 141.58 kg ha-1 was at control.
Published literature revealed that iron application increased the N uptake of plants .Our result was
similar to the findings of [48, 49].
Table 9. Plant Nitrogen (Straw+Grain) uptake as (kgha-1) of mung bean

as affected by different levels of Iron and Zinc
0 113.72 135.02 176.01 141.58 c
5 164.00 190.39 214.82 189.74 b
10 203.30 265.33 323.33 263.99 a
Mean 160.34 c 196.91 b 238.06 a
LSD(0.05) of plant N for Fe= 23.168
LSD(0.05) of plant N for Zn= 32.809
LSD(0.05) of plant N for Fe*Zn= 56.826
Conclusions
From the investigation it was concluded that application of Fe and Zn at the rate of 5 and
10 kg ha-1 respectively significantly enhanced the biological, grain, straw yields, number and weight
of nodules of mung bean. Maximum uptake of nitrogen by the plants straw and grain was also
observed in the plots where Fe and Zn were applied at higher rate of 5 kg ha-1 and 10 kg ha-1
respectively. Such studies are suggested to evaluate the effect of iron and zinc for various
leguminous crops under different agro-ecological zones to improve the nitrogen fixation.
The authors declare that there is no conflict of interest.
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In Vitro Regeneration of Medicinally Important Shrub Carissa opaca

from Shoot Apices and Nodal Segments
Ali Ahmad, Bilal Haider Abbasi, Muhammad Zia*
Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan
*ziachaudhary@gmail.com
Keywords: Shoot Regeneration, Carissa opaca, Medicinal Plant, Cytokinin, MS media, plant
growth regulators, in vitro regeneration
Abstract. The study was aimed to develop efficient shoot regeneration from ex vitro explants of
Carissa opaca, an imperative medicinal reservoir. Shoot apices and nodal segments were inoculated
on MS (Murashige and Skoog) medium containing BAP (6-bezyl amino purine) and Kin (Kinetin)
alone and in combination with NAA (naphthalene acetic acid) and GA3 (Gibberellic acid). Higher
concentrations of both cytokinins were found effective for regeneration from both explants.
However, gibberellic acid and NAA addition with cytokinin, no persuading results were achieved.
The shoot apices were found more effective in in vitro regeneration than nodal segments. The
protocol can be effectively used for in vitro multiplication of C. opaca, genetic transformation, and
secondary metabolite production.
Introduction
Carissa opaca is a perennial shrub comprising of enormous medicinal potential. There are no
reports available on the micropropagation of this medicinally important specie. However, reckless
collection and deforestation have made this specie near to be encompassed in extinct species. There
is an unprecedented need for the reliable source of this plant material for commercial manipulation
either by exploiting modern or conventional plant tissue culture techniques. C. Opaca is locally
known as “Kaurunda” is an evergreen shrub characterized by thorny twigs with alternate leaves. It
is found on relatively high altitude as in Pakistan Kashmir, Murree, Abbottabad and Margalla Hills
are habitat for this plant. The plant is effective in hepatitis and jaundice [1], antipyretic and
anticancer effect [2]. Beside these cardioprotective, restorative potency of reproductive hormones,
hepatoprotective activity and anti-inflammatory activities are associated with the fruits and leaves
while roots are found anti-oxidative in nature [3].
Several plants fail to produce flower or seed and germinate under the influence of some
specific conditions imposed by the climate. This in vitro regeneration technique ensures steady
supply of plants by making use of least space and time [4]. The advantages of In vitro micro
propagation is secondary metabolites production, production of genetically engineered plants,
controlled environmental conditions according to specific plant needs, threatened plant species
conservation, production of clones and identification, year round plant availability, cryopreservation
ensuring the preservation of genetic material and greater multiplication rate and others [5]. The
objective of the present quest was to develop protocol for the in vitro shoot regeneration of Carissa
opaca from explants.

The shoots and nodal explants of Carissa opaca collected from the vicinity of Quaid-i-Azam
University, Islamabad Pakistan were inoculated on MS (Murashige and Skoog 1962 [6]) basal
media supplemented with cytokinin (6-bezyl amino purine; BAP and Kinetin; Kin) alone and with
GA3 (Gibberellic acid) and NAA (naphthalene acetic acid) for shoot organogenesis. Sucrose
(30 g/L) was added in MS media as carbon source and pH of the media was adjusted to 5.7. Nobel
agar 8 g/L was added and 30 ml media was poured in 100 ml conical flask. The flasks were
autoclaved at 121°C and 15 psi for 20 min.
24 Volume 3
Before inoculation of explants, the explants were disinfected by treatment with 0.1% mercuric
chloride solution for 3-4 min and three times washing with autoclaved water. The explants were cut
to a size of 0.5 cm in length and were placed on the media under sterile condition. The flasks were
kept in growth chamber having 25°C and 16/8 day light cycle with light intensity 10000 lux. After
15 and 30 days of inoculation, percentage of shoot regeneration, shoot length, number of shoots and
number of leaves per shoot were noted.
Three explants were inoculated in each flask and three flasks were used for each
concentration. Each value indicates mean ± SD for nine replicates. The means of significantly
different values were compared using Duncan’s multiple range test (DMRT) at P<0.05.
Results
Shoot tip and nodal segments were inoculated on MS media supplemented with BAP (6-
benzyl amino purine) and Kin (Kinetin). Both explants showed 100% regeneration at 4 mg/L BAP
and 3 & 4 mg/L Kin. Shoot tips also showed 100% regeneration at 3 & 5 mg/L BAP and 5 mg/L
Kin. At lower concentrations of BAP, nodal segments showed better response while shoot tip
showed better response at lower concentration of Kin (Fig 1). When both cytokinins were added
with GA3 (Gibberellic acid) or NAA (naphthalene acetic acid), shoot tip explants showed 100 %
regeneration rate at most of the combinations. However 100% response from nodal segments was
observed only on two combinations of Kin and GA3 (Fig 2).
120
Shoot tip Node
100
80
Response (%)
60
40
20
0
0.5 1 1.5 2 3 4 5 0.5 1 1.5 2 3 4 5
BAP Kin Ctr
Concentration (mg/L)
Figure 1. Percentage Growth Response of C. opaca explants on MS supplemented media with BAP
and Kin.
120
Shoot tip Node
100
Response (%)
80
60
40
20
0
3;0.5
4;0.5
3;0.5
4;0.5
3;0.5
4;0.5
3;0.5
4;0.5
3;1
3;2
3;3
4;1
4;2
4;3
3;1
3;2
3;3
4;1
4;2
4;3
3;1
4;1
3;1
4;1
BAP;GA3 Kin;GA3 BAP;NAA Kin;NAA Control
Concentration (mg/L)
Figure 2. Percentage Growth Response of C. opaca explants on MS media supplemented with

cytokinin along withGA3 or NAA.
Table 1 shows that maximum shoot length (1.82 cm) from shoot apices attained at 3 mg/L
while maximum number of shoot (2.8) and maximum number of leaves (4.33) were produce at
4 mg/L BAP. Same concentrations of Kin (3 & 4 mg/L) were found best for shoot length, number
of shoots and number of leaves, respectively (Table 1). At 4mg/L BAP the shoots protruded from
nodal segments attained maximum length (1.17 cm), maximum number of shoots (2.8) and
maximum number of leaves (5.6). Nodal segments also showed good shoot length (1.38 cm) and
number of leaves (4.4) response at 4 mg/L kin however maximum number of shoots (2) were
obtained at 2 mg/L kin (Table 1).
Table 1. Effect of BAP and Kin on shoot regeneration of C. opaca from shoot apices and nodal
segments.
Hormon Shoot Apices Nodal Segment
(mg/L) Length No of Leaves Length No of Leaves
BAP
0.5 0.30±0.07f 1.25±0.43 1.25±0.43c 0.27±0.09c 1.25±0.43c 1.50±0.50cd
1 0.55±0.11e 1.75±0.83c 1.50±0.50c 0.63±0.52bc 2.00±0.71b 3.00±1.58b
1.5 0.23±0.05g 1.33±0.47d 2.00±0.00bc 0.15±0.05d 1.67±0.47bc 3.00±0.82b
2 0.75±0.05d 2.50±0.50ab 2.00±0.0bc 0.20±0.00c 2.50±0.50ab 2.00±0.0c
3 1.82±0.74a 1.50±0.76cd 3.50±1.60ab 0.85±0.11b 1.83±0.69bc 3.80±1.12b
4 1.58±0.29ab 2.80±1.17a 4.33±2.13a 1.17±0.14a 2.80±1.17a 5.60±1.50a
5 1.18±0.21c 1.40±0.49cd 2.50±1.71b 1.20±0.10a 1.40±0.49c 3.20±1.60b
Control 1.35±0.05b 2.00±0.0b 1.50±0.50c 0.15±0.05d 2.00±0.0b 1.50±0.50cd
Kin
0.5 0.40±0.0c 0.50±0.10c 0.50±0.10c 0.50±0.10c 1.00±0.00d 2.00±0.00c
1 0.25±0.05d 0.43±0.05c 0.43±0.05c 0.43±0.05c 1.25±0.43c 2.00±0.00c
1.5 0.20±0.06d 0.10±0.00d 0.10±0.00d 0.10±0.00d 1.60±0.49bc 2.80±0.98b
2 0.35±0.05c 0.60±0.00b 0.60±0.00b 0.60±0.00b 2.00±0.00a 3.00±1.00b
3 1.92±0.20a 1.07±0.35ab 1.07±0.35ab 1.07±0.35ab 1.83±0.37b 4.00±1.15ab
4 1.78±0.29ab 1.38±0.09a 1.38±0.09a 1.38±0.09a 1.80±0.40b 4.40±1.50a
5 1.30±0.24b 1.32±0.26a 1.32±0.26a 1.32±0.26a 1.83±0.37b 4.00±1.15ab
Control 1.35±0.05b 0.15±0.05d 0.15±0.05d 0.15±0.05d 2.00±0.00a 1.50±0.50d
Values are the mean ± standard error (SE) of three replicates per treatment. Each replicate contained four explants.
Values followed by different letters indicate significant a difference between means (P≤ 0.05) according to one way
ANOVA.
26 Volume 3
Maximum number of shoots (2) from shoot apices were also attained when BAP and Kin (3 &
4 mg/L) were combined with different concentrations of GA3 (Table 2). However, shoots did not
attain better length and maximum was observed in control flasks. The combination of BAP
(4 mg/L) or Kin (3 mg/L) with GA3 (3 or 2 mg/L, respectively) were found better to induce leaves
on shoots emerged from shoot apices. Nodal segments also did not show better response in case of
shoot length when cultured on BAP and GA3 combination however 4 mg/L BAP with GA3 (3 or
4 mg/L) was found better for shoot length and number of leaves. The same response was observed
when BAP was replaced with Kin.
Table 2. Effect of cytokinins and GA3 on shoot regeneration of C. opaca from shoot apices and
nodal segments.
Hormone Shoot apices Nodal Segments
(mg/L) Length No of shoots Leaves Length No of shoots Leaves
BAP GA3
3 0.5 0.30±0.10e 1.50±0.50bc 1.50±0.50f 0.80±0.10ab 1.00±0.00b 3.00±1.00b
3 1 0.83±0.04c 1.25±0.43c 2.50±0.87e 0.63±0.05b 1.00±0.00b 2.00±0.00c
3 2 0.80±0.22c 2.00±0.00a 7.33±0.94b 0.30±0.10c 1.50±0.50a 2.00±0.00c
3 3 0.35±0.05e 2.00±0.00a 7.00±1.00b 0.40±0.00c 1.00±0.00b 2.00±0.00c
4 0.5 0.85±0.18c 1.75±0.43b 4.00±0.41c 0.45±0.05c 1.00±0.00b 1.50±0.50c
4 1 1.10±0.10b 1.50±0.50bc 3.50±0.50d 0.77±0.12ab 1.33±0.47a 3.00±0.82b
4 2 0.75±0.15cd 2.00±0.00a 6.50±0.50bc 0.45±0.05c 1.00±0.00b 2.00±0.00
4 3 0.73±0.12cd 1.67±0.47bc 8.67±0.94a 0.40±0.10c 1.50±0.50a 5.00±1.00a
Control 1.35±0.05a 2.00±0.00a 1.50±0.50f 1.00±0.60a 1.00±0.60a 1.80±0.40c
Kin GA3
3 0.5 0.30±0.20d 1.00±0.00d 3.00±1.00c 0.70±0.10b 1.00±0.00b 2.50±0.50cd
3 1 0.60±0.08c 1.67±0.47bc 2.00±0.00d 0.60±0.10c 1.00±0.00b 1.50±0.50d
3 2 0.60±0.08c 2.00±0.82a 8.00±1.63a 0.73±0.05b 1.67±0.47a 5.33±1.89a
3 3 0.64±0.27c 1.80±0.40b 6.40±2.33b 0.30±0.00d 1.00±0.00b 4.00±0.00b
4 0.5 1.45±0.05a 2.00±0.00ab 5.00±1.00bc 1.20±0.10a 1.50±0.50a 4.00±0.00b
4 1 1.00±0.20b 1.00±0.00d 2.00±0.00d 0.95±0.05ab 1.50±0.50a 5.00±1.00ab
4 2 0.50±0.16cd 1.67±0.47bc 6.67±2.49b 0.15±0.05e 1.50±0.50a 3.00±1.00c
4 3 0.75±0.15bc 1.50±0.50c 7.00±1.00ab 0.25±0.05d 1.00±0.00b 3.00±1.00c
Control 1.35±0.05a 2.00±0.00ab 1.50±0.50e 1.00±0.60ab 1.00±0.00b 1.80±0.40d
ANOVA.
Replacement of GA3 with NAA however in combination with BAP or Kin did not much
influences studied parameters. Shoot length did not positively influenced on any combination
however number of shoot and number of leaves got better response at different combination or BAP
or Kin with NAA (Table 3).
Table 3. Effect of cytokinins and NAA on shoot regeneration of C. opaca from shoot apices and
nodal segments.
Hormone Shoot apices Nodal segment
(mg/L) Length No of Leaves Length No of Leaves
BAP NAA
3 0.5 0.76±0.28bc 1.60±0.49cd 6.00±2.53ab 0.18±0.08d 1.25±0.43bc 1.50±0.50b
3 1 0.98±0.13b 1.75±0.43c 7.50±2.18a 0.22±0.16d 1.80±0.40b 2.60±1.74ab
4 0.5 0.65±0.14c 2.17±0.69a 7.33±2.20a 0.35±0.05c 2.50±0.50a 4.00±0.00a
4 1 0.62±0.16c 1.50±0.50d 4.33±1.80b 0.50±0.21b 1.75±0.43b 4.00±2.00a
Control 1.35±0.15a 2.00±0.00b 1.50±0.50c 1.00±0.60a 1.00±0.00c 1.80±0.40b
Kin NAA
3 0.5 0.38±0.19c 1.25±0.43b 5.00±2.24b 0.24±0.10d 1.20±0.40bc 1.80±0.40c
3 1 0.97±0.26b 1.67±0.47ab 6.67±2.40ab 0.47±0.05b 1.33±0.47bc 3.33±0.94bc
4 0.5 0.77±0.23bc 1.50±0.50b 4.50±0.96b 0.35±0.05c 1.50±0.50b 4.00±0.00b
4 1 0.70±0.22bc 1.83±0.37ab 7.50±2.57a 0.47±0.12b 2.33±0.47a 6.00±0.00a
Control 1.35±0.05a 2.00±0.00a 1.50±0.50c 1.00±0.60a 1.00±0.00c 1.80±0.40c
ANOVA.
Discussion
C. opaca shoot tip and nodal explants showed better regeneration at higher concentration of
cytokinin. BAP or kin at 3 mg/L and above showed 100% regeneration potential from shoot tip
explants. When BAP and Kin, were combined with GA3, approximately 100% regeneration
response from shoot tip explant was observed excluding few combinations. Replacing GA3 with
NAA also did not favour the regeneration rate where none of the combinations showed 100%
response for nodal segments while at few combinations all inoculated shoot tips responded well
(100% response). Effectiveness of BAP over Kin for bud break has been reported in a number of
plant species [7, 8]. Profound results of BAP over Kin on in vitro propagation of Caralluma
tuberculata and Cariasa species have been proven by others [9-11].
Limited numbers of shoot buds were obtained due to vigour of shoot buds and inherent nature
of plant. It is evident from results that increasing the concentration of BAP or Kin also triggers
increase in the number of shoots and leaves per plant. Such findings have also been reported by
others. No substantial changes in growth parameters were observed when explants were treated with
cytokinin along with GA3 and NAA. It is evident from others results that gibberellins did not
enhance the shoot length [12]. These results suggest that GA3 was slightly effective than NAA
when used in synergy with BAP or Kin. No significant effect on plant height was observed when
GA3 and NAA were used.
In the node culture, rate of multiplication of C. opaca explants is found less than that which
was brought about through shoot culture in all treatments of plant growth regulators PGRs
(cytokinins, gibberellins and auxins). Shoot cultures have high multiplication rate when cultured at
appropriate medium [13, 14]. As C. opaca is a shrub, the possible reasons for slow growth, dearth
of response to cytokinins, abnormal growth, e.g., hyperhydric transformation, prolonged phenolic
exudation, shoot miniaturization, or stunting, shoot necrosis, or excessive callusing hampering
shoot multiplication optimization in stage II, have been proposed [15] especially when dealing with
woody species.
After 30 days period of shoots establishment, the microshoots were inoculated on full strength
and half strength MS basal media. The inoculated shoots were duped into no roots emergence even
on augmented MS basal media with IAA and IBA (0.25-2 mg/L). The possible reasons could be the
problems like dearth of response (poor or no root induction) to auxin(s), excessive callusing, or
deterioration in overall shoot quality for in vitro micropropagation as Lynch also proposed these
problems for in vitro rooting [16].
28 Volume 3
Conclusions
The present experiments document the very first protocol for C. opaca plantlets’ production
by tissue culture. This study conclude that MS basal media appended with BAP and Kin at 3 and
4 mg/L concentrations was found to be vigorous for shoot regeneration from ex vitro explants.
However, other synthetic hormones like TDZ or woody plant media can be used to achieve
successful rooting and acclimatization. The established in vitro propagation procedure for shoot
proliferation could be used as a substitute for the propagation of C. opaca plantlets.
References
[1] A.M. Abbasi et al., Ethnobotanical study of wound healing herbs among the tribal
communities in Northern Himalaya Ranges District Abbottabad, Pakistan, Pak. J. Bot. 6
(2010) 3747-3753.
[2] S. Sahreen et al., Hepatoprotective effects of methanol extract of Carissa opaca leaves on
CCl 4-induced damage in rat, BMC Com. Alt. Med. 11(1) (2011) 48.
[3] S. Izhar, D. Ahmed, Carissa opaca: A plant with great potential for future drugs for
degenerative and infectious diseases, Chem Select. 1(12) (2016) 3005-3011.
[4] S. Prakash, J. Van Staden, Micropropagation of Hoslundia opposita Vahl – a valuable
medicinal plant, South African J. Bot. 73 (2007) 60-63.
[5] Y. Sidhu, In vitro micropropagation of medicinal plants by tissue culture, The Plymouth
Student Scientist. 4 (2011) 432-449.
[6] T. Murashige, F. Skoog, A revised medium for rapid growth and bio assays with tobacco
tissue cultures, Physiologia Plantarum. 15(3) (1962) 473-497.
[7] M.S. Rathore et al., Micropropagation of elite genotype of Jatropha curcas L. through
enhanced axillary bud proliferation and ex vitro rooting, Biomass and Bioenergy. 83 (2015)
501-510.
[8] A.K. Patel et al., In vitro propagation and ex vitro rooting of Caralluma edulis (Edgew.)
Benth. & Hook. f.: an endemic and endangered edible plant species of the Thar Desert,
Scientia Horticulturae. 165 (2014) 175-180.
[9] R.U. Rehman et al., In vitro propagation of chungah (Caralluma tuberculata NE Brown),
Pak. J. Bot. 46 (2014) 2189-2194.
[10] M.A. Imran et al., Effect of adenine sulphate (ads) with cytokinins on multiple shoot
production in Carissa carandas (L.), International Journal of Pharma and Bio Sciences. 3(1)
(2012) 473-480.
[11] R. Rai, K.K. Misra, Micropropagation of Karonda (Carissa carandas) through shoot
multiplication, Scientia Horticulturae. 103(2) (2005) 227-232.
[12] A.P. Costa et al. Branching, flowering and fruiting of Jatropha curcas treated with ethephon
or benzyladenine and gibberellins, Anais da Academia Brasileira de Ciências. 88 (2016) 989-
998.
[13] A. Varshney, M. Anis, Trees: propagation and conservation: biotechnological approaches for
propagation of a multipurpose tree, Balanites aegyptiaca Del, Springer Science & Business
Media, 2014.
[14] S.N. Hasmah, A. Bhatt, C.L. Keng, Micropropagation of Asam Karanda (Carissa carandas
Linn), Pertanika Journal of Tropical Agricultural Science. 36(1) (2013) 89-98.
[15] E.E. Benson, Sepecial symposium: In vitro plant recalcitrance in vitro plant recalcitrance: An
introduction, In Vitro Cell. Dev. Biol. Plant. 36 (2000) 141-148.
[16] P.T. Lynch, Tissue culture techniques in in vitro plant conservation, in: Plant Conservation
Biotechnology, 2014, pp. 67-68.
Screening of Sunflower Genotypes for Potassium Use Efficiency in

Irrigated Soil Condition
Sobia Baby Jamro, Naheed Akhtar Talpur, Mukesh Kumar Soothar*,
Zial ul Hassan Shah, Mahendar Kumar Sootahar and Ayaz Ali Panhwar
Department of Soil Science, Sindh Agriculture University, Tandojam, Pakistan
*
mukeshksootar@gmail.com
Keywords: Sunflower, genotypes, K use efficiency
Abstract. A field experiment was conducted during summer 2016 to screen out sunflower
(Helianthus annuus L.) genotypes for their potassium (K) use efficiency ratio. Eight sunflower
genotypes were tested; Samsung 20, Mehran 2, Ho-1, Melabour, Samsung 30, Valugur, Chinika and
Sputnik in randomised complete block design (RCBD) with the two treatments comprised of
potassium at 50 and 0 kg K ha-1 along with source SOP recommended dose fertilizer respectively.
The results revealed that the treated and control plots (50 and 0 kg K ha-1) produced different values
for of seeds (1763.1 and 1588.5 head-1), shoot dry weight (23.0 and 19.11 g), head diameter (17.45
and 15.72 cm), seed yields (2065.8 and 1918.7 kg ha-1), seed K % (0.60 and 0.30%) and diagnostic
tissue % (3.54 and 2.65%) respectively. The considerable increase was found in seeds head-1
(10.99%), shoot dry weight (20.35%), head diameter (11.01%), seed yields (11.31%) seed K %
(100%), and leaf K % (33.58%). Among genotypes, Ho-1 was highly effective added K fertilizer.
The more seed (2039.7 head-1), shoot dry weight (25.86 g), head diameter (20.20 cm), seed yields
(2409.5 kg hat-1). Moreover seed K % and leaf K % was also high in variety Ho_1 (0.65% and
(5.05%) respectively. The Ho-1 genotype showed significant response to K fertilizer but the Ho-1
and Chinika were more efficient in utilization of K.
Introduction
Sunflower is a potential crop that can bridge the gap between oil consumption and supply
locally [1]. Sunflower cultivation is well suited to the prevailing cropping as its cultivation does not
replace major crops [2, 3]. The oil received from the sunflower seed is favourable to human health
containing vitamins A, D, E and K soluble vitamins [4, 5, 1]. In Pakistan, sunflower possesses the
potential to overcome with the edible oil deficit and it is well adapted to existing agro-ecological
conditions. There are many factors that are responsible for decline in soil fertility; and inefficient
use of fertilizers is the major constraint for low productivity. Chemical fertilizers play a significant
role in achieving higher crop yields [6]. The application of adequate fertilizers leads to enhance
nutrient uptake in plant and nutrient balance in soil ultimately increasing production of crops [7].
Potassium (K) is an essential nutrient not only for growth and root development of the plant but also
in regulating the function of other macro and micronutrients. Due to potassium inadequacy, the
plant resistance to insect pests, diseases and other natural hazards has been decreased; and
horticultural crops are more at threat of soil borne diseases and other insect pest infestations [8].
The potassium levels of soils in Pakistan and particularly in Sindh province are going to be depleted
day by day due to extensive cropping and high yielding varieties uptake substantially higher
amounts of K to produce high yields [9]. So far there is no natural source to replenish it in to the
soil; however due to its low levels in soils negative effects on crop yield and quality are seen in
some parts of the country [10]. Supplementation of K-fertilizers is muriate of potash and sulphate of
potash, the later one is a good source of potassium but it is very expensive and unfortunately
farmers of Sindh province cannot afford it. Muriate of potash is cheaper source of potassium and
also contains more K as compared to sulphate of potash but unluckily it contains chloride ion,
which already exists in high quantity in soils of Sindh province due to arid and semi-arid climate.
Therefore, with the application of muriate of potash, chloride level in the soil will increase to touch
the climax and this more chloride will damage the crops instead of getting benefit and this may face
heavy economic losses [11]. Sunflower needs K2O form 1.28% or in some cases 3.0 to 6.7% to
produce desired crop yield [12]. It has been reported in study that the current yield level is very low
as compared to the potential yield of many sunflower hybrids. Among various factors responsible
for low yield, management of fertilizers may be of much importance [13]. Many researchers have
observed and recommended that 75-100 kg K ha-1 is profitable in terms of crop growth, high yield,
seed yield, plant height and oil content under normal soil conditions [14-17]. Sivamurugan et al. [18]
found that 40 kg ha-1 K2O increased growth parameters and yield of the sunflower in the summer and
spring seasons, respectively. Amanullah and Khan et al. [19] recommended 100 kg potash along
with 45 kg of phosphorus; while Siddiqui et al. [20] suggested application of 45 kg ha-1 to be the
optimum fertilizer dose for sunflower growth and yield. Asadi [21] reported that the best yield
was found in 200 kg ha-1 level of K2SO4 treatment and the lowest yield was in the 50kg ha-1 of the
KCl treatment.
Keeping in view the rapid mining of K from Pakistani soils, negligible K fertilization and
high K requirement of sunflower, it is hypothesized that the modern high yielding and hybrid
sunflower varieties may require an adequate supply of K-fertilizer to offer optimum yield. This field
study was carried out to evaluate the response of eight sunflower genotypes to apply fertilizer of
potassium for their growth and yield. This study was based on screening of sunflower genotypes for
potassium use efficiency ratio in irrigated soil condition of Tandojam.

The experiment was conducted during 2016-17 at the field of Oilseeds Section, Agriculture
Research Institute Tandojam to evaluate the growth and yield response of eight sunflower
genotypes to potassium fertilizer. The experimental block was divided into three main plots and
further these were divided into eight sub-units of 3.0 m × 5.0 m (15.0 m2) with 03 replications. The
seeds of eight sunflower genotypes; Samsung 20, Mehran-2, Ho-1, Melabour, Samsung 30,
Valugur, Chinik, Sputnik were sown by drilling method in lines and after completion of
germination, thinning was done to maintain 45 cm spacing between plant to plant and 60 cm
spacing between rows.
The soil samples were collected at depths 0-45cm and were brought to the laboratory of Plant
Nutrition, Department of Soil Science, Sindh Agriculture University, Tandojam for analysis of
physico-chemical properties and AB-DTPA extractable K. The collected soil samples were
analysed for EC by EC meter, organic matter by Walky and Black method, pH digital pH meter,
Texture by Bouyoucos Hydrometer method and available K was determined by using flame
photometer.
Leaf/Seed K contents
After harvest, the sunflower leaves and seeds were separately obtained from the experimental
field to determine leaf and seed K content from the treatments of all genotypes. The leaf and seed
samples were determined by wet acid digestion. The samples were digested with nitric perchloric
acid mixture (1:2). The digested samples were analyzed for K by using flame photometer.
K-use efficiency ratio
The K-use efficiency ratio was calculated according to the formula suggested by [22] Brar et al.
Agronomic observations
Following agronomic observations were recorded; seeds per head, head diameter (cm),
shoot dry weight (g) and seeds yield (kg ha-1).
32 Volume 3
Statistical analysis
The data thus collected were statistically analysed using Statistix ver. 8.1. The ANOVA was
derived to examine the significance of treatment effect; while the LSD test was applied to compare
the mean values for assessing significance effect among treatments.
Results
In order to screen out some sunflower genotypes for potassium (K) use efficiency ratio.
Agronomic parameters and soil analysis including leaf and seed K content was examined. In control
plots the genotypes were fertilized with NPK @100-50-0 kg ha-1; while in treatment plots, the
experimental crop was fertilized with NPK @100-50-50 kg ha-1. The experiment revealed the
following results.
Shoot dry weight (g)
The data regarding shoot dry weight (Table-1) shows that the analysis of variance depicted
that the treatment effect on shoot dry weight was significant (P<0.05) and sunflower genotypes also
varied (P<0.05) for shoot dry weight. However, interactive effect of treatments and genotypes was
insignificant on shoot dry weight (P>0.05). The addition of K (100-50-50 kg ha-1 NPK) resulted in
higher shoot dry weight (23.20 g) as compared to 19.11 g under recommended fertilizer application
of 100 kg N and 50 kg ha-1 P (Control). The increase in shoot dry weight in treated plots was
20.35 percent over control. The variety response showed that Ho-1 produced maximum shoot dry
weight (25.86 g), followed by genotypes Mehran 2, Sputnik and Chinika with average shoot dry
weight of 23.49, 22.72 and 20.77 g, respectively. However, the lowest shoot dry weight (18.73 g)
was recorded in genotype Valugur.
Table 1. Shoot dry weight (g) of sunflower genotypes as affected by K fertilizer
Treatments
Mean for
S. No. Genotypes Control Treatment
genotypes
(NPK @100-50-0 kg ha ) (NPK @100-50-50 kg ha-1)
-1
1 Samsung 20 17.64 20.38 19.01DE
2 Mehran 2 20.95 26.33 23.49B

3 Ho-1 23.20 28.53 25.86A
4 Melabour 17.40 20.65 19.03DE
5 Samsung 30 17.64 20.44 19.01DE
6 Valugur 17.15 20.30 18. 73E
7 Chinika 18.84 22.70 20.77CD
8 Sputnik 20.46 24.98 22.72BC
Mean for treatments 19.11B A
23.00 (20.35%) -
Head diameter (cm)

The (Table 2) analysis of variance in regards to sunflower head diameter suggested that
addition of potash fertilizer significantly affect the head diameter (P<0.05); and genotypes effect on
this trait was also significant (P<0.05); while the interactive effect of treatments and genotypes was
insignificant (P>0.05) on head diameter. The results (Table 2) showed that addition of K(50 kg ha-1)
produced sunflower heads of greater diameter (17.45 cm) as compared to (15.72 cm) in control
given recommended fertilizer (100-50-0 kg ha-1 NPK). The increase in head diameter in treatment
plots (100-50-50 kg ha-1 NPK) was 11.01 percent over control. The varietal response indicated that
sunflower variety HO-1 produced heads of maximum diameter (20.20 cm), followed by genotypes
Mehran 2, Sputnik and Chinika with average head diameter of 18.24, 17.46 and 16.74 cm,
respectively. However, the minimum head diameter (14.93 cm) was recorded in genotypes Valugur.
Sunflower genotypes Samsung 20, Melabour and Samsung 30 showed similarity in head diameter
(P>0.05). The interactive effect showed that the maximum head diameter (21.25 cm) was recorded
in variety HO-1 when treated with additional 50 kg K ha-1 while genotype valugur at control
remained lowest in head diameter (14.15 cm).
Table 2. Head diameter (cm) of sunflower genotypes as affected by K fertilizer
Treatments
Mean for
genotypes
(NPK @100-50-0 kg ha-1) (NPK @100-50-50 kg ha-1)
1 Samsung 20 14.20 15.76 14.98D
2 Mehran 2 17.29 19.19 18.24B
3 Ho-1 19.14 21.25 20.20A
4 Melabour 14.36 15.94 15.15D
5 Samsung 30 14.23 15.80 15.02D
6 Valugur 14.15 15.71 14.93D
7 Chinika 15.87 17.62 16.74C
8 Sputnik 16.55 18.37 17.46BC
Mean for treatments 15.72B 17.45A (11.01%) -
Seeds head-1
The (Table 3) statistical analysis describes significant variation in the number of seeds head-1
due to addition of K source (SOP) in addition to recommended fertilizer application (P<0.05) and
genotypes of diversified origin also responded differently to K addition to fertilizer dose (P<0.05),
However, insignificant interactive effect of treatment and genotypes (P>0.05) was recorded. The
treatment comprised of K addition resulted in an increased number of seeds (1080.7 head-1) over
control where the number of seeds declined to (1064.7) head-1. The increase in seeds head-1 in
treatment plots (K addition) was 10.99 percent over control. The varietal response showed that Ho-1
produced maximum number of seeds (1482.0 head-1), followed by genotypes Mehran 2, Sputnik
and Chinika with 1282.7 1208.7 and 1060.7 seed head-1, respectively. However, the lowest number
of seeds (803.0 head-1) was recorded in Valugur. Sunflower genotypes Samsung 20, Melabour, and
Samsung 30 showed similarity (P>0.05) in number of seeds head-1. The interactive effect showed
that the highest number of seeds (1484.0 head-1) was recorded in variety Ho-1 with recommended
fertilizers; while genotypes valugur in control remained least for number of seeds (793.3 head-1).
Table 3. Seeds per head of sunflower genotypes as affected by K fertilizer
Treatments
Mean for
genotypes
(NP@100-50 kg ha-1) (NPK @100-50-50 kg ha-1)
1 Samsung 20 1434.3 1591.9 1513.1E
2 Mehran 2 1746.4 1938.8 1842.6B
3 Ho-1 1933.5 2145.9 2039.7A
4 Melabour 1450.4 1609.9 1530.1DE
5 Samsung 30 1437.9 1596.1 1517.0E
6 Valugur 1429.8 1587.1 1508.5E
7 Chinika 1570.3 1779.7 1675.0CD
8 Sputnik 1705.1 1855.6 1780.3BC
34 Volume 3
Seeds yield (kg ha-1)

The (Table 4) ANOVA related to the data on sunflower seed yield kg ha-1 describes that
application of potash resulted in a significant effect on seed yield kg ha-1 (P<0.05); and sunflower
genotypes included in this experiment responded variably for seeds yield kg ha-1 (P<0.05); whereas
the interactive effect of applied K fertilizer and genotypes was insignificant (P>0.05) on seeds yield
kg ha-1. The data indicated that treatment plots (50 kg K ha-1) produced significantly higher
sunflower seeds yields (2065.8 kg ha-1) as compared to those in control where 1918.7 kg ha-1 seeds
yield was obtained. The treatment plots (50 kg ha-1) increased 11.31 percent of seeds yield kg ha-1
over control. Among the varieties, HO-1 produced higher seeds yield (2473 g kg ha1). However, the
lowest seeds yield (1720g kg ha-1) was achieved in valugur. The treatment interaction indicated that
maximum seeds yield (2573g kg ha-1) was achieved from variety HO-1 with the addition
(50 kg K ha-1); while genotypes valugur remained lowest in seeds yield (1906g kg ha-1).
Table 4. Seeds yield (kg ha-1) of sunflower genotypes as affected by K fertilizer
Treatments
Mean for
genotypes
(NPK @100-50-0 kg ha-1) (NPK @100-50-50 kg ha-1)
1 Samsung 20 1713 1900 1806.5C
2 Mehran 2 2046 2226 2136B
3 Ho-1 2246 2573 2409.5A
4 Melabour 1740 1933 1836.5C
5 Samsung 30 1726 1913 18.19.5C
6 Valugur 1720 1906 1813C
7 Chinika 2133 1886 2009.5B
8 Sputnik 2026 2190 2108B
Leaf K (%)
The (Table 5) analysis of variance for the data on leaf K-uptake showed that the effect of K
addition (NPK 100-50-50 kg ha-1) was significant on leaf K %, and genotypes of diversified origin
also showed varied leaf K %. However, treatment × genotypes interaction was insignificant
(P>0.05) on leaf K %. The results indicated that the addition of K resulted in increased leaf K %
(3.54%) as compared to 2.65% leaf K % determined in crop given control fertilizers. The leaf K %
in treatment plots was 33.58 percent higher over control. The genotypes effect indicated that the
leaf K % was higher in variety Ho-1 (5.05%), followed by genotypes Chinika, Mehran-2 Sputnik
and Melabour with average leaf K-uptake of 3.50, 3.15, 3.15 and 3.00 percent, respectively.
However, the lowest leaf K percent (2.30%) was determined equally in genotypes valugur,
Samsung 30 and Samsung 20. These results indicate that addition of K in the fertilizer was
beneficial for leaf K-uptake of sunflower.
Table 5. Leaf K (%) of sunflower genotypes as affected by K fertilizer

Treatments
Mean for
genotypes
(NPK @100-50-0 kg ha-1) (NPK @100-50-50 kg ha-1)
1 Samsung 20 2.00 2.60 2.30C
2 Mehran 2 2.30 4.00 3.15BC
3 Ho-1 4.70 5.40 5.05A
4 Melabour 3.00 3.00 3.00BC
5 Samsung 30 1.60 3.00 2.30C
6 Valugur 2.00 2.60 2.30C
7 Chinika 3.30 3.70 3.50B
8 Sputnik 2.30 4.00 3.15BC
Seed K (%)
The (Table 6) analysis of variance for the data on seed K-uptake indicated that the effect of K
50 kg K ha-1 was significant on seed K %, while the effect of genotypes and treatment genotype
interaction was also significant on seed K % (P<0.05). The data showed that the addition of K
(50 kg K ha-1) resulted in higher seed K % (0.60%) as compared to 0.30% seed K-uptake recorded
in crop. The seed K % in treatment plots was 100 percent higher over control. The genotypes effect
showed that the seed K-uptake was higher in variety Ho1 (0.65%), followed by genotypes Mehran
2, Sputnik and Chinika with average seed K % of 0.60, 0.55 and 0.45 percent, respectively.
However, the lowest seed K % (0.25%) was determined in genotypes Mehran-2 Samsung 20. The
treatment and genotypes interaction indicated that the highest seed K percentage (0.8%) was
determined equally in varieties Ho-1 and genotypes, Mehran 2 sown under NPK fertilizers dose;
while the minimum seed K percentage (0.1 %) was recorded in genotypes 30, Samsung 20 and
Melabour grown in control. This result indicates that addition of K in the fertilizer was beneficial
for seed K-content of sunflower.
Table 6. Seed K (%) of sunflower genotypes as affected by K fertilizer
Treatments
Mean for
genotypes
(NPK @100-50-0 kg ha-1) (NPK @100-50-50 kg ha-1)
1 Samsung 20 0.1 0.4 0.25G
2 Mehran 2 0.1 0.8 0.25G
3 Ho-1 0.5 0.8 0.65A
4 Melabour 0.1 0.7 0.4E
5 Samsung 30 0.4 0.4 0.4E
6 Valugur 0.3 0.3 0.3F
7 Chinika 0.4 0.7 0.55C
8 Sputnik 0.5 0.7 0.6B
K-use efficiency ratio

The K-use efficiency ratio of different sunflower genotypes with addition of potash at the rate
of 50 kg ha-1 was calculated and statistical analysis (Table 7) suggested a significant (P<0.05)
treatment impact on the K-use efficiency ratio of sunflower genotypes over control. The results
showed that the highest K-use efficiency ratio calculates in kg ha-1 (653kg ha-1) was determined
36 Volume 3
sunflower genotype HO-1, followed by genotypes Chinika, and Melabour, with K-use efficiency
ratio of 493kg ha-1, 386kg ha-1 and 375kg ha-1 respectively. However, the lower K-use efficiency
ratio of 360kg ha-1 and 333kg ha-1 was determined in sunflower genotypes Mehran-2 and Sputnik,
respectively. This indicates that with use of K (50 kg ha-1) all the sunflower tested genotypes
showed significant response to applied K, but variety Ho-1 and genotypes Chinika and Melabour
were more efficient in utilization of applied K.
Table 7. K-use efficiency ratio of sunflower genotypes as affected by potash fertilizer
Treatments
Control Treatment
S. No. Genotypes
(NPK @100-50-0 kg (NPK @100-50-50 kg KUE
ha-1) ha-1)
1 Samsung 20 1713 1900 374C
2 Mehran 2 2046 2226 360B
3 Ho-1 2246 2573 654A
4 Melabour 1740 1933 386C
5 Samsung 30 1726 1913 374C
6 Valugur 1720 1906 372C
7 Chinika 2133 1886 494B
8 Sputnik 2026 2190 328B
Mean for treatments 1918.75B 2065.8A (11.31%)
Discussion
Sunflower genotypes Samsung 20, Mehran 2, Ho-1, Melabour, Samsung 30, valugur, Chinika
and Sputnik were planted under field conditions to screen out for potassium use efficiency ratio and
these varieties were examined for certain agronomic parameters as well as for seed and leaf K
percentage. The treatments were comprised of NPK @100-50-50 kg ha-1; with control (NPK @100-
50-0 kg ha-1). The findings of the study are discussed in this section.
The findings of the study show that sunflower growth and yield performance was
significantly variable due to the addition of K.The treated plots and control plots produced different
values for number of seeds (1763.1 and 1588.5 head-1), shoot dry weight (23.0 and 19.11 g), head
diameter (17.45 and 15.72 cm), seeds yields (2065.8 and 1918.8 kg ha-1), seed K percentage (0.60
and 0.30 and leaf percentage (3.54 and 2.65%). There was a considerable increase in seeds head-1
(10.99%), shoot dry weight (20.35%), head diameter (11.01%), seed yields (11.31%), seed K
percentage (100%), leaf K percentage (33.58%). Among varieties, Ho-1 was highly efficient to
utilize added K level with more shoot dry weight (25.86 g), seeds (2039.7 head-1), head diameter
(20.20 cm), seeds, yields (2409.5g kg ha-1), while seed K % was higher in Ho1 (0.65%) and leaf K
percent was highest in variety Ho-1 (5.05%). Agronomic performance and seed/plant K % of
sunflower significantly improved with addition of potash. The genotypes of diversified origin
responded variably to potash application for agronomic performance and seed/plant K and K-use
efficiency ratio of different sunflower genotypes with addition of potash at the rate of 50 kg ha-1
was significant (P<0.05) impact on the K-use efficiency ratio of sunflower genotypes over control.
The results of the present study are in agreement with those of many past workers who have
worked in different parts of the world on similar aspects. [14, 15, 16] reported that increase in leaf
area, plant height, stem girth, head diameter, number of achenes head-1, 1000-achenes weight,
harvest index and oil content of sunflower, yield with 100 kg K2O ha-1. [17] achieved highest seed
yield of 2.235 t ha-1 by applying 75 kg K ha-1 and 75-100 kg K ha-1 was considered as the optimum
level, beyond this level the application of potash is not profitable. [23] indicated that the highest
1000-seed weight (53.71g) was obtained from the plot fertilized with 150 kg K ha-1, while the
minimum 1000-seed weight (46.41g) was obtained in control plots. [24] recommended 50 kg ha-1 K
in addition to 80 kg N and 50 kg ha-1; while [18] found that K at 40 kg ha-1 gave the highest values
of head diameter (15.3 and 13.6 cm), number of seeds per head (655 and 585), 100-seed weight (5.24
and 5.20 g) and seed yield (1540 and 1313 kg ha) in the summer and SWM season, respectively. In a
similar investigation, [25] reported that 90 kg K ha-1 was found to be most effective K rate in
combination with recommended N and P fertilizers, they suggested that 60 kg K in addition to
120 kg N and 90 kg P ha-1 as optimum fertilizer levels for sunflower crop under the experimental
conditions. [26] achieved highest seed yield (1231.47 kg ha-1) with 130-90-90 kg NPK ha-1
treatment; and from India [27] reported that 120 kg K in addition to 90 kg N ha-1 and 120 kg P2O5
ha-1 were found to be an effective and efficient combination for achieving higher seed yields and oil
content in tested sunflower variety. Amanullah and Khan [19] reported that 100 kg potash along
with 45 kg of phosphorus per hectare was most effective combination of P and K in addition to
uniform rate of nitrogen. [20] proved the incorporation of 90-45-45 kg ha-1 as optimum fertilizer
dose for sunflower growth and yield. Chajjro et al. [11] noted that adequate K nutrition
(120 kg K ha-1) enhanced the growth and achene yield of both sunflower hybrids even under
adequate soil K condition. Mollashahi and Fanaei [28] indicated that use of potassium fertilizers
improves quantitative and qualitative characteristics of sunflower. Memon [29] concluded that the
sunflower crop splendid performance when supplied with N-P fertilizers at the rate of 150-100 kg
ha-1 with highest yield and oil content along with 50 kg ha-1 K in the form of MOP. The study of
[30] showed that maximum seed yield (2813 kg ha-1) was produced by fertilizer combinations of
150:100:100 NPK kg ha-1 and minimum in control plot (1813 kg ha-1). The new genotypes
responded variably to K application for agronomic performance and seed/plant K-uptakes; and
screening of new lines for K use efficiency showed that Ho-1, Mehran-2, Chinika and Sputnik are
most promising genotypes that possess significant potash use efficiency to convert it into increased
seed yield. The K uptake was also markedly higher in genotypes Mehran-2, Chinika and Sputnik
and these genotypes could be the future potential genotype. Similar results have been reported by
many research workers. [24] revealed that K use efficiency of sunflower genotypes varied
significantly and there was no relation of increased K uptake by leaf or seed with the yield level of
genotypes. [31] reported that the K application had significant impact yield attributes of sunflower
genotypes and local genotypes showed simultaneously development in K use efficiency and
increased yield compared to the hybrids. [25] reported that application of potash to sunflower had
significant impact on crop performance, but the varietal response to K application levels varied
considerably. There was no linear relationship of K uptakes with the seed yields. [28] grown a set of
sunflower cultivars and after screening test it was observed that (cv. MSFH-8) variety responded
120 kg K positively with increasing seed yield and plant uptakes. [21] applied K2SO4 at the rate of
200 kg ha-1 in addition to N and P at the recommended levels for particular soils and found that
sunflower varieties responded positively to increased K rates upto 100 kg ha-1, further increase in K
results adverse effect on the crop yields and plant uptakes. Most of the hybrids responded positively
to increased K rates, but K up to 100 kg ha-1 could be beneficially applied, because further increase
in K resulted in negative effects on yield of all the varieties tested. [30] used sunflower hybrids (SF-
187 and Parsun-1) against various K levels and found varied response of sunflower hybrids to
increased K levels, However, a combination of 150:100:100 kg ha-1 NPK resulted in optimistic
results regarding agronomic performance and plant nutrient uptakes. [32] evaluated the impact of
potassium use efficiency of sunflower hybrids and found that Helio 251 had higher potassium use
efficiency as compared to rest of the hybrids.
Conclusion
The present study has been concluded that the sunflower genotypes performed the best against
potassium fertilizer dose. It was observed that variety HO-1, or genotypes Chinika and Melabour
are most promising genotypes that possess significant potash use efficiency ratio to convert it into
increased seed yield.
38 Volume 3
References
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in controlled conditions, Desert. 12 (2008) 99-104.
[2] M.S.A. Ahmad et al., Photosynthetic performance of two mungbean (Vigna radiata) cultivars
under lead and copper stress, Int. J. Agric. Biol. 10 (2009) 167–172.
[3] V.S. Rathore, R.C. Gautam, S.K. Kaushik, Yield, quality and nutrient uptake by sunflower as
influenced by weed and nutrient management, J. Annals Agric. Res. 22(3) (2001) 443-444.
[4] M.K. Hussain, E. Rasul, S.K. Ali, Growth analysis of sunflower under drought conditions,
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sunflower conditions, Agric. Natural Res. Sci. 2 (1998) 31-43.
[6] T.I. Hussain, M.A. Ahmed, EM Technology-A new looks for IPNM, in: Proc. Symp.,
Integrated Plant Nutrient Management, NFDC, Islamabad, Pakistan, 2000.
[7] J.A. Adediran et al., Application of organic and inorganic fertilizer for sustainable maize and
cowpea yields in Nigeria, J. Plant Nut. 27(7) (2004) 1163-1181.
[8] M.I. Bajwa, F. Rehman, Soil and fertilizer potassium, in: A. Rashid, K.S. Memon (Eds.) Soil
Science, National Book Foundation, Islamabad, 1996, pp. 317-338.
[9] S.M. Mian et al., Effect of MOP and SOP on plant chloride uptake and soil properties in a
rice-wheat rotation, Pak. J. Soil Sci. 14(1-2) (1998) 70-74.
[10] I. Cakmak, The role of potassium in alleviating detrimental effects of abiotic stresses in
plants, J. Plant Nutr. Soil Sci. 168 (2005) 521-530.
[11] M.A. Chajjro et al., Sunflower hybrids differentially accumulate potassium for growth and
achene yield, Pak. J. Agri., Agril. Engg., Vet. Sci. 29(1) (2013) 31-43.
[12] D.J. Reuter, J.B. Robinson, Plant analysis: An Interpretation Manual, 2nd Indian Ed., SBS
Publishers & Distributors Pvt. Ltd., New Delhi, India, 2008.
[13] M. Aslam et al., Effect of single and combined use of various organic amendments on wheat
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[14] R. Ahmad, Physio-Agronomic characteristics of semi dwarf and standard height sunflower
hybrids as affected by nutritional area and potassium application, Ph.D. thesis, Department of
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[15] M.T. Ramzan, Effect of different doses of potash on yield and oil content of sunflower, MSc.
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[16] A. Pervaiz, Effect of different sources and levels of potash on yield and oil content of spring
sunflower (Helianthus annuus L.), M.Sc. Thesis, Dept. Agron., Univ. Agri. Faisalabad, 1996.
[17] A.U. Chaudhry, M. Mushtaq, Optimization of Potassium in sunflower, Pak. J. Biol. Sci. 2(3)
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[18] A.P. Sivamurugan, A. Balasubramanian, C.R. Chinnamuthu, Effect of N, P and K levels and
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(Helianthus annuus L.), Pak. J. of Biol. Sci. 2(2) (1999) 402-403.
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Sex Determination in Nutmeg Seedlings Using Scar Primers

Francis Dadzie Mintah
Kwame Nkrumah' University of Science and Technology, Ghana
kofimintahfm@yahoo.com
Keywords: Nutmeg, SCAR primers, sex determination
Abstract. Myristica fragrans H. is a dioecious plant with male and female flowers on different
trees. At the juvenile stage of nutmeg, their sexes is not morphologically identified until at the
flowering stage. However, the female is more economical than the male plants. This challenge is
making the work of nutmeg farmers difficult and also inhibiting the development of the spice and
flavouring industry in Ghana. Hence, the use of molecular technique to characterize male and
female nutmeg plants. SCAR markers are easy, specific, and reliable and have a high sense of
reproducibility than other markers. Two different primers (F-Napf-76, R-Napf-77 and F-Napf-70,
R-Napf-71) were used for the PCR amplification. It was revealed that the total genomic DNA of
samples from both mature plant and seedlings of nutmeg were of good quality and was much
concentrated by giving good DNA bands. Although there were no distinctions among the bands,
they all lie at the same level (0.3kbp) indicating that the samples were of similar molecular weight.
However, double PCR amplification was not seen in all samples when run on a 1% agarose gel but
single amplifications were observed.
Introduction
Myristica fragrans H. is a dioecious plant with male and female flowers on different trees
although hermaphrodite flowers and bisexual trees occur rarely. It produces two separate spices,
namely nutmeg and mace. Nutmeg is the dried kernel of the seed and the mace is the dried aril
surrounding the seed [1]. Nutmeg is indigineous to Mollucas Islands (Indonesia) and thrives well in
warm humid conditions in locations with annual rainfall of 150cm and more [1]. However, it is
currently produced in many places such as India, Malaysia, various Caribbean islands, New Guinea,
and Africa; to be specific Ghana which was introduced in the 1960’s by the Moluccas. The nutmeg
plant is noted to be propagated by two different methods: seedling and vegetative. Gestation period
is more than a month and it takes approximately twenty (20) years to bear mature fruits then after
that it bears fruit every year and production rate increases after ten years. World production of
nutmeg is estimated to an average between 10,000 and 12,000 tonnes (9,800 and 12,000 long tons;
11,000 and 13,000 short tons) per year, with annual world demand estimated at 9,000 tonnes
(8,900 long tons; 9,900 short tons); production of mace is estimated at 1,500 to 2,000 tonnes (1,500
to 2,000 long tons; 1,700 to 2,200 short tons) [2]. In Ghana, the Plant Genetic Resource Research
Institute of the Council for Scientific and Industrial Research (Bunso) harvested about
96,136 nutmeg fruits in the year 2012 and about 15,017 nutmeg fruits in the year 2013.The
economic importance of nutmeg is enormous. The fruit has proven to be very useful in the
preparation of brain tonic, for skin care, as flavoring additive, for pain relief, liver and kidney
detoxifier, sleep aid and for bad breath treatment [3]. Nutmeg is safe in lesser quantities but toxic in
higher quantities [4]. Nutmeg causes a wide adverse reaction to the nervous system and
gastrointestinal tissues. It also causes rapid heart rate and dehydration [5]. However, in its freshly-
ground form, nutmeg contains myristicin, a monoamine oxidase inhibitor and psychoactive
substance which can induce convulsions, palpitations, nausea, eventual dehydration, and
generalized body pain [6].
An important problem that farmers face in the production of nutmeg is that the sex
determination of nutmeg is not revealed morphologically at the juvenile stage until the flowering
stage, making it difficult to obtain an appropriate ratio of males and females plants to be planted on
the field. Although there has been a lot of approach in determining the sexes of nutmeg at the
seedling stage for example; leaf venation, genetic tester etc. but has still not been the best [1]. This
problem is inhibiting the development of the spice and flavoring industry in Ghana. In order to
solve such problem, molecular identification techniques using DNA marker is to be applied rather
than a traditional discrimination method that is carried out with comparisons of morphological
traits. Therefore, SCAR markers were used because they are easy, specific, and reliable and have a
high sense of reproducibility than other markers. SCAR markers have been developed for sex
determination in alien crops [7, 8]. Parasnis et al. [9] developed a male-specific SCAR marker in
papaya by cloning a male-specific RAPD (831 bp) fragment and designing longer primers. Lee et
al. [10] discriminated among the genetic polymorphisms of the Korean ginseng cultivar, P.
quinquefolius, and P. notoginseng through the use of SCAR markers. Manoj et al., [11]
distinguished between the male and female Piper longum through the development of SCAR
primers MPS1A and MPS1B. Vinod et al. [12] reported that OPO-08 RAPD marker amplified a
1263 bp band in male which was absent in females of Pandanus fascicularis. A SCAR marker
(MSSRF-01) was designed for this fragment and continued to amplify the specific allele in all the
male plants. Southern hybridization results also confirmed the RAPD work and strongly suggested
that (MSSRF-01) is male specific molecular marker in Pandanus fascicularis. Devaiah and
Venkatasubramanian [13] developed a SCAR marker for authentication of Pureria tuberosa. OPA
08600 bp marker specific to P. tuberosa. This RAPD amplicon was converted to the SCAR marker
which revealed the expected amplicon (320 bp) in male plants. However, by increasing the
specificity, the results are less sensitive to changes in reaction conditions and are more reproducible
[14]. Consequently, in order to avoid wastage of labour, time and resources, assurance of yield
outcome, prevent contamination from unintended species or trees confronting farmers the purpose
of the study was to identify the sexes of nutmeg seedlings using SCAR markers, and facilitate better
management of nurseries to obtain a higher proportion of females to males seedlings of nutmeg to
be planted in the field.
Literature Review
Sex determination
In the animal kingdom, the separation of female and male functions among individuals is the
common sexual system. In contrast, most flowering plants are hermaphroditic, with flowers bearing
both female and male reproductive organs. Only about 6% of angiosperms are dioecious, where
individuals either produce staminate or pistillate flowers [15]. In a minority of dioecious plants, sex
determination depends on sex chromosomes, usually an XY system, in which males are
heterogametic (XY) and females are homogametic [16-18]. According to Matsunaga and Kawano
[18], two types of sex chromosomes exist in nature homomorphic sex chromosomes, in which the
sex chromosomes are morphologically indistinguishable from autosomes, and heteromorphic sex
chromosomes, which can be discriminated in cytological analyses. Undoubtedly, Heteromorphic
sex chromosomes have been reported in several families (e.g., Cannabis and Humulus,
Cannabinaceae; Silene, Caryo-phyllaceae) [18], but our understanding of their evolution and
genetics is still relatively poor [19].
Conventional Sex diagnostics
Differences between clones and closely related species are not always absolute using
morphological characters [20] and distinguishing between clonal and seedling progenies are
impossible due to very limited morphological variations in this crop [21]. However, several
attempts made for identification of sex of nutmeg seedlings based on morphological and chemical
characters [21] reported only limited successes [22]. Phadnis and Choudhary [23] advocated a
colorimetric test that they claimed could distinguish sex in approximately two thirds of seedlings
while Nayar et al. [24] examining the epidermal cells of nutmeg leaf described the different
structures of calcium oxalate crystals in seedlings of male and female plants. In a preliminary study,
it was indicated that characteristics such as leaf shape, essential oil content and composition and
42 Volume 3
profile of phenolics differ in male and female plants [25]. Contrarily, a study conducted by
Mezencev et al. [26] confirmed the absence of obvious relationship between the extents of
morphological variations and that of changes at the DNA level already reported in rice. Therefore,
earlier studies suggested the use of similar molecular techniques for identification in case of a
confusion or misidentification of genotypes where not much of a morphological variation
whatsoever exist [27].
Molecular sex diagnostics
SCAR markers were developed for sex determination in alien crops [7,8]. However extensive
studies involving sex pooled DNA samples from more number of individuals and primers are
needed to confirm this. Therefore, for molecular markers linked to specific genes there is a need for
screening large number of RAPD primers [28] which is usually more than 50bp [22]. A high
potential of RAPD markers in detecting molecular polymorphism between individuals of an
accession [29], phenotypically pure stocks [30] and intra varietal variations among donor plants
[26] have been demonstrated. Interestingly, Lemos et al. [31] identified a sex specific primer
marker in papaya and a PSDM was isolated. It is likely that these markers are linked to sex
determining loci, though the sex determining mechanism is not yet characterized in nutmeg.
Nevertheless, female specific bands were isolated by RAPD in Myristica [32]. Furthermore, Lee et
al. [10] conducted a study to develop the easier discrimination method, which could demonstrate
genetic polymorphisms among the Korean ginseng cultivar, P. quinquefolius, and P. notoginseng
by using ISSR marker technique and conversion of polymorphic ISSR markers to SCAR markers.

Source of plant materials
Twenty (20) seedlings of nutmeg of six (6) months old together with leaves from known sex
mature nutmeg plants were taken from a nutmeg plantation in Bunso which is located in the Eastern
Region of Ghana.
Reaction Mix Preparation and Concentrations
The Accupower reaction mixture for PCR contained 8μl master mix (containing the premix,
both male specific and gender neutral primers dissolved in distilled water), and 2μl of extracted
total genomic DNA, raising the working volume of the PCR reaction mix to 10μl for each tube.
Gel preparation
A 1% agarose gel was used for running of both the genomic and the PCR samples as well.
Agarose gel was used rather than polyacrylamide gel because of its ability to hold the DNA
fragments within the gel without running into the electrolyte containing TAE buffer.
DNA extraction and gel electrophoresis
Extraction of total genomic of DNA of the leaf samples was done using the CTAB protocol
developed by Takrama [33]. A 1% agarose gel was used for running of both the genomic and the
PCR samples as well. Portions of the extracted genomic DNA were loaded onto the agarose gel and
subjected to electricity. The gel together with the electrolyte and the electricity works with the
principle that small fragments of DNA run down the gel faster than larger ones. Gel electrophoresis
of the total genomic DNA was done to check the quality or integrity of the DNA. DNA isolates
were subjected to PCR and PCR products were loaded on the gel to check variation and
polymorphism.
Table 1. Sequence of Primers Used

PRIMER DESIGNATION SEQUENCE SIZE (bp)
1 F-Napf-76 5’-GAGGATCCCTATTAGTGTAAG-3’ 21
R-Napf-77 5’-GAGGATCCCTTTTGCACTCTG-3’ 21
2 F-Napf-70 5’GGATCCCTATTAG-3’ 15
R-Napf-71 5’-GAGGATCCCTTTTGC-3’ 15
Source: Parasnis et al., [9]
Table 2. Optimized PCR parameters
STEP TEMPERATURE (⁰ C) DURATION
Initial Denaturation 95 3 minutes
Denaturation 95 1 minutes
Annealing 55(primer 1) 45(primer 2) 2 minute 40 cycles
Extension 72 7 minutes
Final Extension 72 5 minutes
Results
DNA Integrity test
DNA quality check showed that good quality DNA was extracted from all leaf samples as
shown in Fig. 1 below. It clearly shows that the total genomic DNA of samples from both mature
plant and seedlings of nutmeg were of good quality and was much concentrated by giving good
DNA bands. Although there were no distinctions among the bands, they all lie at the same level
(0.3kbp) indicating that the samples were of similar molecular weight.
Figure 1. Extracted genomic DNA integrity test. Lane L 1kbp ladder, lane M1-F2 matured male
and female, lane 1-20 contains seedlings
PCR amplification
DNA fragments appeared clearly after running the genomic DNA with the Accupower premix
using the PCR cycling parameter. Fig. 2 below demonstrated that DNA fragments from the mature
males are of the same kilo base pairs (0.40kbp) lying at the same level but those from the mature
44 Volume 3
female are of different base pairs. However, Lanes 4, 5, and 6 contained DNA samples from an
assumed male, assumed female, and assumed male respectively but lie at the same level
corresponding to the mature male sample but do not correspond to the marker. Interestingly, there
was single amplification for all samples.
Figure 2. Separated PCR products of extracted genomic DNA on a 1% agarose gel (Primer 1). L is
1kbp marker, M1-F2 contained mature male and female respectively, lane 1-20 contained seedlings
Figure 3. Separated PCR products of extracted genomic DNA on a 1% agarose gel (Primer 2). L is
1kbp marker, M1-F2 contained mature male and female respectively, lane 1-20 contained seedlings
Discussion
Primer 1 & 2 were male-specific which should bind unto all samples which are male (both
mature and assumed male) leaving all female samples (both mature female and assumed female)
unbind to distinctively show that those with the same kilo base pairs (kbp) with the marker are
males and those which are different in kilo base pairs (kbp) with the markers as females. Primer 3 is
a gene neutral (neither male nor female specific) which should bind to all samples. However, the
primers used were designed for pawpaw not for nutmeg, although they are all dioecious plants [9].
Parasnis et al., [34] who conducted similar experiment on pawpaw using the same primers had
similar results. Sex determination in nutmeg has been worked on in previous experiments by using
different primers thus the OPA (Operon) and they yielded good results. A number of polymorphic
loci observed were 20 with a total of 160 making the mean number of polymorphic band/ primer.
All the primers showed monomorphic loci, the maximum given by OPA 01 primer [22]. However,
the primers OPK 01 and OPD 15 which were identified as male specific markers for date palm and
rumex respectively [35, 36] have given polymorphism for nutmeg with female specificity. The sex
determination system in date palm and rumex was XX-XY (XX –female; XY- male) chromosome
system [37]. In nutmeg also it appears that sex is genetically determined with a strong possibility of
a mono-factorial sex-determining mechanism [38]. Single amplifications show that there is genetic
difference between nutmeg and pawpaw. The genetic mechanisms involved in dioecy vary in
different plants [39]. Monomorphic loci was identified whiles other studies identified polymorphic
loci. Previous experiments in dioecious plants proved that among 100 primers (OPC) evaluated, 75
produced clear DNA profile yielding a total of 971 amplification bands for the bulked sample of
male, female and hermaphrodite papaya plants of which 89 bands were polymorphic [39]. This can
be attributed to the MgCl2 concentration, temperature and duration for the PCR. According to
Sheeja et al. [40] the appropriate concentration of MgCl2 for effective primer amplification is
2.0 ng but in this experiment, 0.5 ng was used. Moreover, the concentration of DNA samples is a
contributing factor to polymorphism or monomorphism.
Conclusion
Monomorphic loci is common for all nutmeg plants. Hence the inability of farmers to
differentiate between a male or female juvenile nutmeg plant when grown on the field.
Optimization of PCR parameters will enhance the tendency of achieving polymorphism as
identified in other dioecious plants. A further designed nutmeg specific primers could have a better
potential of showing unique polymorphism.
The author declares that there is no conflict of interest.
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46 Volume 3
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Keyword Index
C
Cadmium 1 S
Carissa opaca 23 SCAR Primers 40
Chromium 1 Sex Determination 40
Copper 1 Shoot Regeneration 23
Crude Oil 1 Sunflower 30
Cucurbita maxima 1
Cytokinin 23 Z
Zinc 1
F Zn 13
Fe 13
G
Genotypes 30
H
Heavy Metals 1
I
In Vitro Regeneration 23
K
K Use Efficiency 30
L
Lead 1
M
Medicinal Plant 23
MS Media 23
Mung Bean 13
N
Nodules 13
Nutmeg 40
P
Plant Growth Regulators 23
R
Rhizophagus irregularis 1
Author Index
A
Abbasi, B.H. 23
Ahmad, A. 23
E
Eneh, G.D.O. 1
F
Fawad, M. 13
J
Jamal, A. 13
Jamro, S.B. 30
K
Khan, M.I. 13
M
Mintah, F.D. 40
O
Okon , O.G. 1
Okon, J.E. 1
P
Panhwar, A.A. 30
S
Shah, Z.U.H. 30
Sootahar, M.K. 30, 30
T
Talpur, N.A. 30
Tariq, M. 13
Z
Zia, M. 23

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Arbuscular Mycorrhizal Fungi (AMF) as Buffer for Heavy Metals Phytoextraction by

Arbuscular Mycorrhizal Fungi (AMF) as Buffer for Heavy Metals

Materials and Methods

Sources of Crude Oil and Experimental Soil

Results and Discussion

Figure 3. Mycorrhizal Colonization of C. maxima in Soil Contaminated with Crude Oil

Response of Mung Bean Crop to Different Levels

Keywords: Mung bean, Nodules, Fe, Zn.

Materials and Methods

Results and Discussions

Grain Yield (kg ha-1) 800

Table 9. Plant Nitrogen (Straw+Grain) uptake as (kgha-1) of mung bean

[10] P. Balachander, P. Nagarajan, S. Gunasekaran, Effect of organic amendments and

In Vitro Regeneration of Medicinally Important Shrub Carissa opaca

Materials and Methods

Figure 2. Percentage Growth Response of C. opaca explants on MS media supplemented with

Screening of Sunflower Genotypes for Potassium Use Efficiency in

Keywords: Sunflower, genotypes, K use efficiency

Materials and Methods

1 Samsung 20 17.64 20.38 19.01DE

2 Mehran 2 20.95 26.33 23.49B

Head diameter (cm)

Seeds yield (kg ha-1)

Table 5. Leaf K (%) of sunflower genotypes as affected by K fertilizer

K-use efficiency ratio

Sex Determination in Nutmeg Seedlings Using Scar Primers

Keywords: Nutmeg, SCAR primers, sex determination

Materials and Methods

Table 1. Sequence of Primers Used

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