Journal of the Saudi Society of Agricultural Sciences (2016) 15, 171178

King Saud University

Journal of the Saudi Society of Agricultural Sciences

www.ksu.edu.sa
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FULL LENGTH ARTICLE

Eect of nitrogen on growth, nutrient assimilation,

essential oil content, yield and quality attributes in
Zingiber ocinale Rosc.
Minu Singh *, M. Masroor A. Khan, M. Naeem

Plant Physiology Section, Department of Botany, Aligarh Muslim University, Aligarh 202002, India

Received 28 June 2014; revised 9 November 2014; accepted 10 November 2014

Available online 18 November 2014

KEYWORDS Abstract A simple randomized pot experiment was conducted to determine the effect of different
Nitrogen; concentrations of nitrogen (N) on growth, biochemical, quality and yield attributes of Zingiber
Rhizome protein; ofcinale Rosc. a medicinally important spice plant. Plants were supplied with ve levels of basal
Essential oil; nitrogen viz. 0 (Control), 40, 80, 120 and 160 kg N per ha. The performance of the crop was
Rhizome yield; adjudged in terms of various parameters viz. plant height, number of leaves, fresh and dry weights
Zingiber ofcinale Rosc. of shoot and rhizome, total chlorophyll content, leaf and rhizome NPK content, rhizome carbohy-
drate and protein content, and analysed at 120 and 180 days after planting (DAP). The data
revealed that nitrogen application signicantly stimulated most of the studied attributes. Of the ve
N concentrations, 120 kg N per ha soil proved optimum for most of the parameters studied as com-
pared to the control at both crop stages.
2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction for atulence and colic. Ginger possesses antioxidant proper-

ties and also has edible oils and fats to protect them against
Ginger (Zingiber ofcinale Rosc.), a member of the Zingibera- oxidative rancidity (Ippoushi et al., 2003). It also exerts antit-
ceae family is used as a broad spectrum antiemetic. It is valued umorigenic and immunomodulatory effects and is an effective
as herbal medicine for a number of conditions including those antimicrobial and antiviral agent (Reddy and Lokesh, 1992;
affecting the digestive tract, headache, nausea, vomiting and Vimala et al., 1999; Akoachere et al., 2002). Though, India is
motion sickness (Dedov et al., 2002; Borrelli et al., 2004). a major producer of ginger, the growing on supply demand
Use of ginger has become a great vogue as a household remedy owing to its nutritive and therapeutic values is putting a heavy
strain in the market. In the coming years, the demand may
* Corresponding author. Mobile: +91 9412176810. even exceed supply. One of the best alternatives to counter this
E-mail address: [email protected] (M. Singh). adverse situation is the cultivation of this plant on scientic
Peer review under responsibility of King Saud University. lines. This would augment the yield and quality of ginger
ensuring its steady supply in market.
Mineral nutrients applied basally or through foliar applica-
tion, enhance the plant productivity and could be used as tools
Production and hosting by Elsevier to ameliorate the quality of herbal medicines. As far as
http://dx.doi.org/10.1016/j.jssas.2014.11.002
1658-077X 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
172 M. Singh et al.

fertilizers are concerned judicious use of nitrogenous fertilizer blotting sheets. Later, the plant height was measured, number
is more important. Nitrogen (N) is the single most important of leaves and tillers per plant was counted and leaf length and
limiting nutrient for crop growth and hence ample N availabil- breadth were measured. Fresh weight of the plant parts was
ity acts as a key contributing factor towards wholesome recorded using electronic balance. Dry weight of plant parts
improvement in agriculture production (Singh, 2009). It is con- was recorded after drying them in a hot air oven at 60 C.
sidered to be of prime importance as it plays several important
roles in metabolic and regulatory processes in plants. 2.5. Yield analysis
Realizing the immense medicinal importance of ginger
crop, there is a dire need to improve the yield potential of The yield attributes, including number of primary and second-
the crop under the local environmental conditions. Keeping ary ngers per plant and fresh rhizome yields per plant were
all these points in view, a research framework was made with recorded at harvest (240 DAP).
the purpose to evaluate the effect of different nitrogen levels on
ginger, under the agro-ecological conditions at Aligarh, U.P. 2.6. Biochemical and quality parameters

2. Materials and methods 2.6.1. Estimation of chlorophyll content

Total chlorophyll content in fresh leaves was estimated using
2.1. Soil analysis
the method of Lichtenthaler and Buschmann (2001). The fresh
tissues from interveinal leaf area were ground in a mortar and
Before planting the crop, the experimental pots were lled with pestle containing 80% acetone. The optical density (O.D.) of
5.0 kg homogenous mixture of soil and farmyard manure (4:1). the solution was read at 663 and 645 nm (chlorophyll a & b)
The soil was maintained at proper moisture to ensure better using a spectrophotometer (Shimadzu UV1700, Japan). Photo-
germination of rhizomes. Physico-chemical characteristics of synthetic pigments were expressed as mg g 1 FW.
the soil were: texture-sandy loam, pH (1:2) 6.9, E.C. (1:2)
0.46 m mhos per cm, and available N, P and K are 84.82, 2.6.2. Determination of N, P and K content in leaves and
9.63 and 144.08 mg per kg of soil, respectively. The soil sam- rhizomes
ples were tested at the Government Soil Testing Laboratory, Leaf and rhizome samples from each treatment pot were
Quarsi Farm, Aligarh. A uniform basal dose of phosphorus digested for the estimation of N, P and K content. 100 mg of
(P) and potassium (K) was also given basally. oven dried powder of leaves and rhizomes was carefully trans-
ferred to a digestion tube and 2 cm3 of AR grade concentrated
2.2. Plant material sulphuric acid was added. It was heated on a temperature con-
trolled assembly at 80 C for about 2 h. On heating, the con-
Mother rhizomes of ginger (3035 g), free from the visible tents of the tube turned black. It was cooled for about
nematode or fungal infection were cut into pieces of uniform 15 min at room temperature and then 0.5 cm3 of 30% hydro-
size, with each of the pieces having a sound bud. Before plant- gen peroxide (H2O2) was added drop by drop. The addition
ing the seed pieces were surface sterilized with 95% ethyl alco- of H2O2 followed by gentle heating was repeated until the con-
hol. Later, they were washed thoroughly with distilled water tents of the tube turned colourless. The aliquot (peroxide-
and sown in earthen pots (25 cm diameter 25 cm height) at digested material) was used to estimate percentage content of
a depth of about 10 cm. N, P and K in the leaves and rhizomes on dry weight basis.

2.3. Experimental design 2.6.3. Estimation of nitrogen

The N content of the sample was estimated according to the
A pot culture experiment was conducted in this regard in the method of Lindner (1944) with slight modication by
natural conditions of net-house in the Department of Botany, Novozamsky et al. (1983). One hundred milligram of the leaf
A.M.U., Aligarh (27.88N latitude, 78.08E longitude and powder was digested. A 10 cm3 aliquot (peroxide-digested
187.45 m altitude). The experiment was conducted according material) was transferred to a 50 cm3 volumetric ask. To it,
to the simple randomized design, using ve basal levels of 2 cm3 of 2.5 N sodium hydroxide and 1 cm3 of 10% sodium sil-
nitrogen, viz. 0 (control), 40 80 120 and160 kg N per ha i.e. 0 icate solutions were added to neutralize the excess of acid and
(Control), 18, 36, 54 and 72 mg N per kg soil applied as urea. to prevent the turbidity, respectively. In a 10 cm3 graduated
N was applied in 3 splits at 45, 90 and 135 days after planting test tube, 5 cm3 of this solution was taken and 0.5 mL Nesslers
(DAP). A recommended uniform basal dose of P22K42 was reagent was added. The O.D. of the solution was recorded at
also applied at the time of planting the crop (Handbook of 525 nm, using the spectrophotometer. The N content was esti-
Agriculture, 2002). Each treatment was replicated three times. mated using a standard graph prepared by graded dilutions of
The plants were kept free from weeds and watered as and when ammonium sulphate.
required.
2.6.4. Estimation of phosphorus
2.4. Growth analysis The method of Fiske and Subba Row (1925) with slight mod-
ication by Rorison et al. (1993) was used to estimate the
At 120 and 180 DAP, three plants from each treatment were phosphorus content in the digested material of leaves and rhi-
uprooted carefully and washed with tap water to wipe off all zomes. A 5 cm3 aliquot was taken in a10 cm3 graduated test
adhering particles. They were surface-dried thereafter using tube. To it, 1 cm3 molybdic acid (2.5%) was added carefully,
Effect of nitrogen on quality attributes in Zingiber ofcinale Rosc. 173

followed by addition of 0.4 cm3 of 1-amino-2-naphthol-4-sul- 2.6.9. Estimation of essential oil content
phonic acid. When the colour turned blue, the volume was The essential oil content from ginger was extracted by using
made up to 10 cm3 with double distilled water (DDW). The Clevenger method. The samples of the powdered rhizomes
O.D. of the solution was recorded at 620 nm using the spectro- were steam distilled for three hours using Clevengers appara-
photometer. The P content was estimated using a standard tus and the essential oil was extracted and determined gravi-
graph prepared by graded dilutions of monopotassium metrically (Clevenger, 1928).
phosphate.
2.7. Statistical analysis
2.6.5. Estimation of potassium
Potassium contents in the leaves and rhizomes were deter- Data were analysed statistically using a one way ANOVA and
mined ame photometrically (Hald, 1946) in the peroxide- F-values were calculated accordingly. Fishers Least signicant
digested material with the help of emission spectra using spe- difference (LSD) was calculated at 5% level of probability
cic lters. In the ame-photometer, the solution (peroxide according to Gomez and Gomez (1984) and Duncans Multi-
digested material) was discharged through an atomizer in the ple Range Test (DMRT) was employed to separate means
form of a ne mist into a chamber, where it was drawn into using different letters in the tables and gures.
a ame. Combustion of the elements produced the light of a
particular wavelength [(k max for K = 767 nm (violet)]. The 3. Results and discussion
light produced was conducted through the appropriate lters
to impinge upon a photoelectric cell that activated a galva-
nometer, resulting in the digital reading of the respective In the present study, basal application of N proved signi-
samples. cantly effective on various attributes studied at different
growth stages. Among the applied doses of nitrogen, N120
proved best by causing maximum stimulation.
2.6.6. Estimation of rhizome carbohydrate content
The carbohydrate content of the samples was analysed by the 3.1. Growth attributes
method of Yih and Clark (1965). One hundred mg of dried rhi-
zome powder was transferred to a boiling tube containing Table 1 shows that basal application of N improved all the
5 cm3 of 2.5 N HCl. The content was hydrolysed by keeping growth characteristics at both the growth stages (120 and
it in a boiling waterbath for 3 h and its volume was made up 180 DAP). Application of N120 proved optimum and elevated
to 100 cm3 with DDW. The sample was then centrifuged at the plant height, number of leaves per plant and number of til-
4000 rpm for 10 min and the supernatant was collected. lers per plant by 40.8%, 52.7% and 71.7% at 120 DAP and
1 cm3 of this extract (supernatant) was taken in a test tube, 32.8%, 39.5% and 105.5% at 180 DAP, respectively (Table 1).
to which 1 cm3 of 5% distilled phenol was added. Afterwards, Correspondingly, an enhancement in shoot fresh weight, rhi-
the test tube was placed in chilled water and 5 mL of concen- zome fresh weight, shoot dry weight and rhizome dry weight
trated H2SO4 was added. The O.D. of the content was mea- was recorded at similar sampling stages. The promoting effect
sured at 490 nm using the spectrophotometer. Rhizome of N on these growth parameters can be explained on the basis
carbohydrate content was estimated using standard graph pre- of the fact that N supply increases the number of meristematic
pared by graded dilutions of glucose. cells and their growth leading to the formation of shoots (til-
lers) in addition to leaf expansion and number (Lawlor,
2.6.7. Estimation of rhizome protein content 2002). It also acts as a key component of a number of metab-
The protein content in rhizome was estimated by the method olites, including amino acids, chlorophylls, co-enzymes,
of Lowry et al. (1951). The rhizome powder (50 mg) was trans- enzymes, proteins, purines and pyrimidines (Salisbury and
ferred to a mortar, adding 1 cm3 of 5% cold Trichloroacetic Ross, 1992; Lea and Guadry, 2001; Marschner, 2002). Fur-
acid (TCA). To it 0.5 cm3 of Folin phenol reagent was added thermore, N application is known to increase the levels of cyto-
rapidly with immediate mixing. The content turned blue. It kinin, which affects cell wall extensibility (Arnold et al., 2006).
was left for 30 min for maximum colour development. It is, therefore, logical to speculate that N was involved directly
Extracted protein was measured at 660 nm using the spectro- or indirectly in the enlargement and division of new cells and
photometer. Rhizome protein content was estimated using a production of tissues which in turn were responsible for
standard graph prepared by graded dilutions of bovine serum increase in growth characteristics. The benecial effect of
albumin. soil-applied N corroborates the ndings of Xu and Xu
(1999) and Haque et al. (2007a) on ginger crop.
2.6.8. Estimation of oleoresin
3.2. Biochemical and quality attributes
Oleoresin was extracted from the, dried and powdered rhizome
in acetone (ASTA, 1978). 10 g of the sample was weighed and
transferred to a glass column. The contents were exposed to 3.2.1. Total chlorophyll content
(50 cm3) acetone overnight. The ltrate extracted through Regarding the effect of nitrogen, it was noted that all nitrogen
non absorbent cotton was collected in a pre-weighed 100 cm3 treatments increased the total chlorophyll content gradually
beaker. Column was washed with 20 cm3 of acetone. The over the control (N0). Application of N120 proved optimum
extracts were pooled and evaporated to dry at 80 C over a at both stages of growth. Of the ve levels of nitrogen, treat-
water bath. The amount of oleoresin was estimated ment N120 showed an increase of 29.9% and 23.7% over the
gravimetrically. control at 120 and 180 DAP, respectively (Fig. 1). Such a
174 M. Singh et al.

Table 1 Effect of ve basal levels on (N0, N40, N80, N120 and N160) on growth attributes of ginger (Zingiber ofcinale Rosc.) at 120
and 180 DAP. Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test.
The data shown are means of three replicates.
Attributes DAP Treatments (kg N per ha soil) LSD at 5%
N0 N40 N80 N120 N160
Plant height (cm) 120 27.43e 30.73d 34.10c 38.63a 37.20a 1.81
180 51.60e 55.87d 61.37c 68.53a 66.40a 3.62
Number of leaves per plant 120 36.00e 41.67d 47.33c 55.00a 51.33b 2.41
180 95.33e 107.00d 119.67c 133.00a 126.33b 4.17
Number of tillers per plant 120 2.33d 2.33d 2.67c 4.00a 3.67b 0.68
180 6.00e 8.67d 10.00c 12.33a 11.33b 0.68
Shoot fresh weight per plant (g) 120 20.51e 24.16d 28.47c 35.90a 31.82b 2.74
180 62.45e 70.32d 83.94c 97.85a 91.20b 5.36
Rhizome fresh weight per plant (g) 120 31.12e 36.73d 43.46c 52.84a 47.33b 3.21
180 77.53e 86.20d 100.47c 118.86a 112.14b 5.82
Shoot dry weight per plant (g) 120 3.96e 4.75d 5.52c 7.43a 6.81b 0.54
180 11.87e 13.44d 16.13c 19.95a 18.39b 1.27
Rhizome dry weight per plant (g) 120 4.81e 5.94d 7.09c 8.72a 8.15b 0.73
180 13.34e 15.09d 17.73c 21.64a 19.80b 1.42

2.5 4
120 DAP 120 DAP
180 DAP a a 180 DAP a b
c
2.0 b d
Chlorophyll content (mg g FW)

c 3
a e

Leaf nitrogen content (%)

d b c a b
-1

c d
d e
1.5
e
2

1.0

1
0.5

0.0 0
1.2 120 DAP 120 DAP 4
180 DAP a b 180 DAP a b
b d c
c e
1.0 d
Leaf phosphorus content (%)

Leaf potassium content (%)

a b a a 3
d c
c b
0.8 d
e

0.6 2

0.4
1
0.2

0.0 0
N0 N40 N80 N120 N160 N0 N40 N80 N120 N160

Nitrogen (kg N per ha) Nitrogen (kg N per ha)

Figure 1 Effect of ve concentrations of nitrogen (0, 40, 80, 120, 160 kg N per ha) on total chlorophyll content, leaf N, P and K contents
of ginger (Zingiber ofcinale Rosc.) at 120 and 180 DAP. Different letters above the bars indicate statistically different means at p 6 0.05.
LSD values are presented at top of the bars.

response of the plants to the basal application of nitrogen is stromal and thylakoid proteins in leaves (Hikosaka, 2004;
expected because N markedly promotes the formation of Cooke et al., 2005; Teixeira et al., 2011). The positive effect
active photosynthetic pigments through increased amounts of of N supply on the formation of chloroplasts during leaf
Effect of nitrogen on quality attributes in Zingiber ofcinale Rosc. 175

growth has also been reported by Hong et al. (2012). In turn, tion throughout the rhizomes. These results agree with the
the chloroplast formation leads to an increase in the lipid con- ndings of Thakur and Sharma (1997), and Majumdar et al.
tent of leaves and chloroplast constituents such as chlorophyll (2002). Furthermore, in the present study, leaf NPK contents,
and carotene. declined with the increase in age of the plants. This could be
attributed to the well known dilution with growth effect
3.2.2. Nutrient elements (Marschner, 2002).
A progressive increase in leaf and rhizome NPK content was
noted from control to N120 that proved optimum at both 3.2.3. Rhizome carbohydrate and protein content
stages. The percentage increase in leaf-NPK content resulted Application of nitrogen on rhizome carbohydrate and protein
from the application of treatment N120 was 18.2%, 15.5% contents was found effective over the control at both stages of
and 8.9% at 120 and 16.5%, 14.3% and 8.4% at 180 DAP, crop growth. Of the ve levels of nitrogen treatments, N120
respectively over the control (Fig. 1). Moreover, the applica- proved superior and signicantly gave maximum values for
tion of optimum dosage of N considerably enhanced the rhi- both the contents. Treatment N120 enhanced the rhizome car-
zome N, P and K content over the control by 22.4%, 12.5% bohydrate and protein contents by 15.3% and 29.5% at
and 7.4% at 120 DAP and by 24.8%, 16.9% and 9.6% at 120 DAP and by 13.6% and 34.1% at 180 DAP, respectively
180 DAP, respectively (Fig. 2). The reason for the benecial relative to the untreated control (Fig. 3). The benecial effect
effect of nitrogen application on the nutrient content in com- of nitrogen on carbohydrate and protein contents of rhizome
parison with the control is obvious. As no nitrogen was may be traced to its various roles in plants. Moreover, the
applied to the control plants, they had to depend totally on interdependence of N supply and carbohydrate utilization
the nutrient present in the soil. On the other hand, treated can be understood in terms of the metabolic requirements of
plants got an adequate supply of this nutrient, ensuring ready N assimilation (Noggle and Fritz, 1986). The enhancing effect
availability and continuous absorption by roots followed by of nitrogen on rhizome protein content might be attributed to
smooth translocation to the foliage and satisfactory distribu- the direct role of N in protein formation (Taiz and Zeiger,

1.0 1.0
120 DAP 120 DAP
180 DAP a b 180 DAP a b
d c d c
0.8

Rhizome phosphorus content (%)

0.8 e e
Rhizome nitrogen content (%)

a a b
c b b
0.6 d c 0.6
e d

0.4 0.4

0.2 0.2

0.0 0.0
2.5 N0 N40 N120 N160
120 DAP N80
180 DAP
a Nitrogen (kg N per ha)
b
Rhizome potassium content (%)

2.0 d c
e b a a
c
d

1.5

1.0

0.5

0.0
N0 N40 N80 N120 N160

Nitrogen (kg N per ha)

Figure 2 Effect of ve concentrations of nitrogen (0, 40, 80, 120, 160 kg N per ha) on rhizome N, P and K contents of ginger (Zingiber
ofcinale Rosc.) at 120 and 180 DAP. Different letters above the bars indicate statistically different means at p 6 0.05. LSD values are
presented at top of the bars.
176 M. Singh et al.

60 12
120 DAP 120 DAP
180 DAP 180 DAP a
50 b 10
Rhizome carbohydrate content (%)

a a c a

Rhizome protein content (%)

b b d b
c c
40 a a e 8
b d
c e
d
30 6

20 4

10 2

0 0
N0 N40 N80 N120 N160 N0 N40 N80 N120 N160

Nitrogen (kg N per ha) Nitrogen (kg N per ha)

3.5
180 DAP
a
3.0 240 DAP b b
c
d
Essential oil content (%)

2.5

2.0 a b
c b
d
1.5

1.0

0.5

0.0
N0 N40 N80 N120 N160

Nitrogen (kg N per ha)

Figure 3 Effect of ve concentrations of nitrogen (0, 40, 80, 120, 160 kg N per ha) on rhizome carbohydrate and protein contents of
ginger (Zingiber ofcinale Rosc.) at 120 and 180 DAP and essential oil content at 180 and 240 DAP. Different letters above the bars
indicate statistically different means at p 6 0.05. LSD values are presented at top of the bars.

Table 2 Effect of ve basal levels on (N0, N40, N80, N120 and N160) on yield attributes of ginger (Zingiber ofcinale Rosc.) at 240 DAP.
Different letters within the same row indicate mean values statistically different at p 6 0.05 as determined by LSD test. The data shown
are means of three replicates.
Attributes Treatments (kg N per ha soil) LSD at 5%
N0 N40 N80 N120 N160
Primary ngers per plant 3.3c 3.7c 4.3b 6.0a 5.7a 0.7
Secondary ngers per plant 6.3d 8.0c 9.7b 11.3a 10.0b 0.7
Rhizome yield per plant (g) 123.7e 147.9d 162.5c 178.3a 170.6b 6.3

2006) and also its favourable effect on the accumulation of sis of some hormones (gibberellins, auxins and cytokinins)
NPK nutrients coupled with their cumulative effect on involved in protein synthesis (Jones et al., 1991) that might
metabolism related to protein synthesis in rhizome. N also have presumably played a role in amelioration of protein
has an inuence on the ribosome structure and the biosynthe- content.
Effect of nitrogen on quality attributes in Zingiber ofcinale Rosc. 177

3.2.4. Essential oil content Ashraf, M., Ali, Q., Iqbal, Z., 2006. Effect of nitrogen application rate
on the content and composition of oil, essential oil and minerals in
The perusal of data revealed that treatment of N120 proved
black cumin (Nigella sativa L.) seeds. J. Sci. Food Agric. 86, 871876.
superior to the other levels of nitrogen and gave maximum val- ASTA, 1978. Ofcial analytical methods of the American Spice Trade
ues for essential oil content at both stages of crop growth. The Association, second ed. American Spice Trade Association Inc,
percentage increase in essential oil content resulted from the New Jersey.
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