G. 2112  
Page 1  
Global Research journal of Natural Science  
& Technology (GRJNST)  
Volume: 04 - Issue 4 (2026), 2112  
ISSN P: 2790-7643 ISSN E: 2790-7651  
www.grjnst.net  
https://doi.org/10.53762/grjnst.04.04.03  
Multi-Location Evaluation of Copper Fertilization on Cane Yield of  
Sugarcane (Saccharum officinarum L.) under Alkaline-Calcareous Soils of  
Southern Punjab, Pakistan  
Received: 15 April 2026. Accepted: 20 May 2026. Published: 20 June 2026.  
Muhammad Javid Qamar (Corresponding Author)  
Soil Fertility (Field), Bahawalpur, Punjab, Pakistan  
javedqamarswt@gmail.com  
https://orcid.org/0009-0005-7271-0807  
Muhammad Rashid Farooq  
Soil Fertility (Field), Bahawalpur, Punjab, Pakistan  
https://orcid.org/0009-0005-3121-7397  
Sehrish Jameel  
Soil and Water Testing Laboratory, Bahawalnagar, Punjab, Pakistan  
https://orcid.org/0009-0003-2983-9336  
Muhammad Bilal  
Soil and Water Testing Laboratory, Dera Ghazi Khan, Punjab, Pakistan  
https://orcid.org/0009-0003-6512-9446  
Aftab Ahmad Sheikh  
Soil and Water Testing Laboratory, Dera Ghazi Khan, Punjab, Pakistan  
https://orcid.org/0009-0003-8306-6565  
Beenish Butt  
Soil and Water Testing Laboratory for Research, Multan, Punjab, Pakistan  
https://orcid.org/0009-0000-0741-6875  
Muhammad Asif  
Soil and Water Testing Laboratory, Khanewal, Punjab, Pakistan  
https://orcid.org/0009-0008-9139-7870  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
Copyright © 2026 GRJNST. This article is published under an Open Access model. It is made available to the public under the terms of the Creative  
Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use and distribution  
G. 2112  
Page 2  
Tahir Akbar  
Soil and Water Testing Laboratory, Muzaffargarh, Punjab, Pakistan  
https://orcid.org/0009-0003-1699-0256  
Ali Rizwan  
Soil and Water Testing Laboratory, Bahawalnagar, Punjab, Pakistan  
https://orcid.org/0000-0002-8248-8016  
Samina Hamid  
Soil and Water Testing Laboratory, Rawalpindi, Punjab, Pakistan  
https://orcid.org/0000-0002-0510-4138  
Muhammad Imran  
Soil and Water Testing Laboratory, Rahim Yar Khan, Punjab, Pakistan  
https://orcid.org/0009-0007-2965-0612  
Muhammad Asadullah  
Soil and Water Testing Laboratory, Bahawalnagar, Punjab, Pakistan  
https://orcid.org/0009-0008-0391-2466  
Abstract  
Sugarcane (Saccharum officinarum L.) productivity in southern Punjab is  
constrained not only by macronutrient imbalance but increasingly by latent  
micronutrient deficiencies on the region's alkaline-calcareous soils, where free  
CaCO₃ and chronically low organic matter immobilize plant-available copper. A  
multi-location field trial was therefore conducted during the 2023–24 crop  
season at three contrasting sites of the Bahawalpur–Bahawalnagar tract ;  
Haroonabad-1, Haroonabad-2, and Bahawalpur; to evaluate the response of  
spring-planted CPF-246 cane to graded levels of broadcast copper sulphate  
pentahydrate (CuSO₄·5H₂O). Five treatments comprising 0, 6, 8, 10, and 12 kg  
CuSO₄·5H₂O ha⁻¹, applied over a uniform NPK basal of 250–175–125 kg  
ha⁻¹, were arranged in a two-factor randomized complete block design with  
three replications at each site. Soil analysis confirmed alkaline pH (8.22–8.40),  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 3  
low organic matter (0.23–0.86%), and contrasting EC (1.31–2.9 dS m⁻¹) and  
macronutrient status across the three sites. Combined analysis of variance  
revealed highly significant effects of treatment (F = 14.34, p < 0.01) and  
location (F = 46.36, p < 0.05) on cane yield, while their interaction was  
marginally non-significant (F = 2.29, p = 0.0825). Cu fertilization  
progressively improved cane yield, with pooled mean yields rising from  
104,993.8 kg ha⁻¹ in the unfertilized control to 112,843.2 kg ha⁻¹ at the  
highest tested dose. The greatest cane yield response over the NPK-only control  
was recorded at 12 kg CuSO₄·5H₂O ha⁻¹ (+7,849.4 kg ha⁻¹; +7.5%), which  
was therefore identified as the recommended Cu rate for spring-planted  
sugarcane on the alkaline-calcareous soils of southern Punjab. The inclusion of  
12 kg CuSO₄·5H₂O ha⁻¹ as a basal soil-broadcast input, alongside the standard  
NPK schedule, is recommended to capture the latent Cu limitation on cane  
productivity in this agro-ecological zone.  
Keywords: Sugarcane, copper fertilization, CuSO₄, multi-location trial, alkaline-  
calcareous soils  
1. Introduction  
Sugarcane (Saccharum officinarum L.) ranks among the most economically important  
industrial crops of the tropics and subtropics, supplying approximately 80% of the  
world's sugar and a rising share of bioethanol, bagasse-based bioenergy, and allied by-  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 4  
products (FAO, 2023). The crop is cultivated on about 26 million hectares globally,  
with Brazil, India, China, Thailand, and Pakistan jointly accounting for the most of  
world production (FAOSTAT, 2023). In Pakistan, sugarcane occupies roughly 1.19  
million hectares and contributes nearly 2.7% to the value added in agriculture and about  
0.5% to the national GDP, sustaining an extensive network of sugar mills, growers, and  
downstream industries (GoP, 2024). Punjab alone accounts for more than 60% of the  
national cane area, with the Bahawalpur–Bahawalnagar belt of southern Punjab emerging  
as a major cane-producing tract under semi-arid, canal-irrigated conditions (Afghan et  
al., 2024). Despite this scale, the national average cane yield of about 68–74 t ha⁻¹  
remains well below the genetic potential of cultivated varieties and considerably lower  
than yields achieved in comparable agro-ecologies of India and Egypt (Shahzad et al.,  
2019). The persistent yield gap has been linked to imbalanced fertilization, poor varietal  
usage by farmers, marginal land allocation, and, increasingly, to micronutrient  
deficiencies that remain largely overlooked in routine fertilizer programmes (Majeed et  
al., 2022; Manzoor et al., 2023).  
The soils of southern Punjab are characteristically alkaline, calcareous, and low in  
organic matter, with pH values commonly ranging from 7.8 to 8.5 and free CaCO₃  
contents that fix and immobilize several essential micronutrients (Rashid and Ryan,  
2004; Imran et al., 2020). Decades of intensive cropping under canal irrigation,  
combined with NPK-centric fertilizer schedules, have progressively depleted the  
available pools of zinc, boron, iron, and copper across the Indus Basin (Zia et al., 2007;  
Majeed et al., 2022). National soil surveys have consistently reported that more than  
half of Pakistan's cultivated soils are deficient in one or more micronutrients, with  
copper deficiency being particularly common in coarse-textured, low-organic-matter  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 5  
soils of the southern districts (Manzoor et al., 2023). While the agronomic significance  
of zinc and boron has received substantial research attention in cereals and cotton, the  
role of copper in sugarcane productivity under these conditions remains comparatively  
under-investigated.  
Copper is a structural and catalytic constituent of more than thirty plant enzymes,  
including plastocyanin, cytochrome c oxidase, superoxide dismutase, and polyphenol  
oxidase, and is therefore central to photosynthetic electron transport, mitochondrial  
respiration, lignin biosynthesis, and antioxidant defense (Yruela, 2009; Printz et al.,  
2016). Adequate Cu nutrition has been linked to greater chlorophyll stability, stronger  
stalk lignification, improved resistance to lodging, and enhanced sucrose accumulation in  
graminaceous crops (Adrees et al., 2015; Hunter et al., 2023). In sugarcane specifically,  
copper participates in tiller initiation, internodal elongation, disease resistance and the  
activity of enzymes regulating sucrose phosphate synthase, all of which directly govern  
cane stand density and final yield (McCray et al., 2010; Majeed et al., 2022). Field  
studies on Cu-deficient soils have shown that adequate Cu supply increases the number  
of millable canes per unit area and reduces premature tiller mortality, thereby translating  
into measurable yield gains (Naga Madhuri et al., 2013). Deficiency symptoms, often  
masked under field conditions, typically appear as chlorotic young leaves, reduced  
tillering, delayed maturity, and weak stalks that lodge under irrigation or wind stress.  
Despite this physiological centrality, calibrated soil-applied Cu dose–response data for  
sugarcane on alkaline-calcareous soils of Pakistan are sparse, and most current fertilizer  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 6  
recommendations issued by provincial agriculture departments still do not include  
copper as a routine input.  
The few regional studies that have addressed copper nutrition in sugarcane have  
generally been confined to single sites or used a single Cu rate, making it difficult to  
identify an agronomically optimal dose or to determine whether the response is  
consistent across soils of differing fertility status (Naga Madhuri et al., 2013; Majeed et  
al., 2022). A multi-location, dose–response evaluation of copper fertilization in  
sugarcane under the agro-ecological conditions of southern Punjab is therefore  
warranted. The present study was designed (i) to evaluate the response of sugarcane (cv.  
CPF-246) to four graded levels of soil-broadcast copper sulphate pentahydrate  
(CuSO₄·5H₂O), applied over a uniform NPK basal dose, in terms of cane yield during  
the 2023–24 crop season at three contrasting sites of the Bahawalpur–Bahawalnagar  
tract; (ii) to identify the Cu rate that produces the highest yield response over the  
unfertilized control; and (iii) to test whether the Cu response interacts significantly with  
location, thereby clarifying whether a single regional recommendation is justified or  
whether site-specific calibration is required.  
2. Materials and Methods  
2.1 Experimental sites  
The field experiment was conducted during the 2023–24 sugarcane crop season at three  
sites located in the canal-irrigated, semi-arid tract of southern Punjab, Pakistan: two sites  
in District Bahawalnagar (Haroonabad-1 and Haroonabad-2) and one site at  
Bahawalpur. The region is characterized by hot, dry summers, mild winters, low to  
moderate annual rainfall, and predominantly alluvial, calcareous soils developed on Indus  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 7  
Basin sediments. Prior to land preparation, composite soil samples were collected from  
each site at two depths (0–15 cm and 15–30 cm) following standard auger sampling  
protocols (Ryan et al., 2001). Samples were air-dried, ground, sieved through a 2 mm  
screen, and analyzed for electrical conductivity of the saturation extract (ECₑ) by  
conductivity meter, pH (1:1 soil-to-water suspension) by glass electrode, organic matter  
by the Walkley–Black wet oxidation method, available phosphorus by the AB-DTPA  
extraction procedure, and exchangeable potassium by ammonium acetate extraction  
followed by flame photometry (Ryan et al., 2001; Estefan et al., 2013). The soil  
analytical data for the three experimental sites are presented in Table 1.  
All three sites carried alkaline, low-organic-matter soils typical of the southern Punjab  
cropping plain. Haroonabad-1 and Haroonabad-2 represented moderately saline  
conditions (ECₑ 2.4–2.9 dS m⁻¹) with adequate available K (125–177 ppm) and low-  
to-medium available P (6.2–10.9 ppm), while Bahawalpur represented a non-saline (ECₑ  
1.1–1.3 dS m⁻¹) but markedly P- and K-deficient soil (P 3.95–5.71 ppm; K 51–90  
ppm). Organic matter content was uniformly low across all sites (0.23–0.86%), and  
surface pH ranged from 8.22 to 8.40, conditions known to depress the plant availability  
of soil-bound copper.  
Table 1. Soil physico-chemical properties of the three experimental sites at two sampling  
depths.  
Soil parameter  
EC (dS  
pH  
OM  
(%)  
Available P  
(ppm)  
Available K  
(ppm)  
m⁻¹)  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 8  
Soil parameter  
EC (dS  
pH  
OM  
(%)  
Available P  
(ppm)  
Available K  
(ppm)  
m⁻¹)  
Haroonabad-1  
Depth 0–15 cm  
2.5  
2.4  
8.2  
8.1  
0.56  
0.86  
6.3  
6.2  
165  
177  
Depth 15–30  
cm  
Haroonabad-2  
Depth 0–15 cm  
2.9  
2.7  
8.4  
8.3  
0.71  
0.77  
10.9  
9.1  
125  
177  
Depth 15–30  
cm  
Bahawalpur  
Depth 0–15 cm  
1.31  
1.10  
8.24  
8.22  
0.51  
0.23  
5.71  
3.95  
90  
51  
Depth 15–30  
cm  
2.2 Plant material  
A commercial sugarcane variety, CPF-246, was used at all three locations. CPF-246 is a  
widely cultivated, mid-maturing variety recommended for the southern Punjab cane belt  
and was selected for its consistent performance under the prevailing semi-arid, canal-  
irrigated conditions of the region. Disease-free, healthy three-budded setts were obtained  
from certified seed stock and used for planting at all three sites.  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 9  
2.3 Treatments and experimental design  
The experiment evaluated five treatments comprising a graded series of copper sulphate  
pentahydrate (CuSO₄·5H₂O) rates applied over a uniform NPK basal dose. The  
treatments were: T1 = Control (NPK only, 0 kg Cu ha⁻¹); T2 = NPK + 6 kg  
CuSO₄·5H₂O ha⁻¹; T3 = NPK + 8 kg CuSO₄·5H₂O ha⁻¹; T4 = NPK + 10 kg  
CuSO₄·5H₂O ha⁻¹; and T5 = NPK + 12 kg CuSO₄·5H₂O ha⁻¹. The uniform basal  
NPK dose comprised 250 kg N, 175 kg P₂O₅, and 125 kg K₂O ha⁻¹, applied as urea,  
di-ammonium phosphate (DAP), and sulphate of potash (SOP), respectively. Treatment  
codes and nutrient compositions are summarized in Table 2.  
The experiment was laid out as a two-factor randomized complete block design  
(RCBD) with locations and Cu fertilization levels as the two factors, with three  
replications at each site. All five treatments were randomly allocated to plots within each  
replication at each location, and the same randomization protocol was followed at all  
three sites.  
Table 2. Treatment compositions used to study the response of sugarcane to graded  
levels of copper fertilization.  
Treatment  
Code  
N (kg ha⁻¹) P₂O₅ (kg ha⁻¹) K₂O (kg ha⁻¹) CuSO₄·5H₂O (kg  
ha⁻¹)  
T1_Control  
T1  
250  
175  
125  
0
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 10  
T2_Cu_6  
T3_Cu_8  
T4_Cu_10  
T5_Cu_12  
T2  
T3  
T4  
T5  
250  
250  
250  
250  
175  
175  
175  
175  
125  
125  
125  
125  
6
8
10  
12  
2.4 Crop management  
Planting was carried out during the recommended spring planting window of 2023 at all  
three locations. The full doses of P₂O₅, K₂O, and CuSO₄·5H₂O were broadcast  
uniformly over the prepared seedbed and incorporated into the surface soil through a  
final shallow tillage operation immediately before sett placement. The total N dose was  
applied in three equal splits: one-third at planting, one-third at the tillering stage  
(approximately 60–90 days after planting), and one-third at the grand growth stage  
(approximately 120–150 days after planting). Setts were placed end-to-end in opened  
furrows at the recommended depth and covered with moist soil. Irrigation was applied  
through the canal supply system; the first irrigation was given immediately after planting  
to ensure uniform sett germination, followed by subsequent irrigations as required by  
crop stage, evaporative demand, and rainfall. Weeds were controlled by a combination  
of pre-emergence herbicide application and two manual hoeings during the early tillering  
phase. Plant protection measures against borers and sucking pests were applied  
uniformly across all plots whenever scouting indicated treatment thresholds had been  
reached. All other agronomic and plant protection operations were carried out as per the  
standard production recommendations of the Punjab Agriculture Department for  
spring-planted sugarcane.  
2.5 Data recording  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 11  
At physiological maturity, cane yield was estimated by harvesting all canes from the net  
plot area, weighing the harvested produce in the field using a calibrated spring balance,  
and extrapolating the plot yield to a per-hectare basis (kg ha⁻¹). All measurements were  
taken on the net plot area, excluding border rows, to minimize edge effects.  
2.6 Statistical analysis  
The data were subjected to two-factor analysis of variance (ANOVA) appropriate for a  
multi-location randomized complete block design, with locations and treatments treated  
as fixed effects and replications nested within locations. Treatment means were separated  
using Fisher's protected least significant difference (LSD) test at the 5% probability  
level (Steel et al., 1997). The significance of the location, treatment, and location ×  
treatment interaction effects was tested at p ≤ 0.05 and p ≤ 0.01 levels. The yield  
response of each Cu treatment over the unfertilized control was computed for each  
location as the absolute and percentage yield difference from T1. Statistical analyses  
were performed using Statistix 8.1, and graphical presentations were prepared using  
R/RStudio and Microsoft Excel.  
3. Results and Discussion  
3.1 Soil status of the experimental sites  
Soil analytical data (Table 1) confirmed that the three experimental sites represented  
contrasting fertility profiles within the broadly similar alkaline-calcareous matrix of  
southern Punjab. Surface soil pH varied within a narrow range (8.22–8.40) across all  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 12  
three sites, conditions that strongly limit the plant availability of soil-bound copper  
through carbonate fixation and adsorption onto calcium-rich exchange surfaces.  
Electrical conductivity differentiated the sites more sharply: Haroonabad-1 and  
Haroonabad-2 carried moderately saline surface soils (ECₑ 2.5 and 2.9 dS m⁻¹,  
respectively), while Bahawalpur was non-saline (ECₑ 1.31 dS m⁻¹). Organic matter  
content was uniformly low (0.23–0.86%), with Bahawalpur registering the lowest  
surface OM (0.51%) and subsurface OM (0.23%). Available phosphorus ranged from  
3.95 to 10.9 ppm and was clearly inadequate at Bahawalpur, while available potassium  
varied from 51 ppm (deficient, Bahawalpur subsoil) to 177 ppm (adequate, Haroonabad  
subsoils). Collectively, these soil conditions; alkaline pH, low organic matter, and  
variable but generally limiting macronutrient status, represent the broader fertility  
constraints under which the cane crop was raised and were expected to amplify the  
agronomic value of micronutrient supplementation.  
These results align closely with the wider characterization of the Indus Basin cane belt,  
where alkaline pH and free CaCO₃ are recognized as the primary chemical drivers of  
micronutrient immobilization (Rashid and Ryan, 2004; Imran et al., 2020). The low  
organic matter content recorded across all three sites is particularly relevant for copper  
nutrition, because soil organic matter is a principal repository of complexed Cu²⁺  
available for slow release into the soil solution; its depletion under continuous, NPK-  
centric cropping systems is a well-documented driver of latent Cu deficiency on  
Pakistani soils (Zia et al., 2007; Majeed et al., 2022). The site-to-site contrast in salinity  
and macronutrient status further provided a useful natural gradient against which the  
consistency of Cu response could be tested.  
3.2 Analysis of variance for cane yield  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 13  
The combined analysis of variance for cane yield across the three locations revealed  
highly significant effects of both location and treatment, while their interaction was  
marginally non-significant (Table 3). The location effect was significant at the 5%  
probability level (F = 46.36, p = 0.0209), indicating that the three sites produced  
significantly different mean cane yields, an expected outcome given their contrasting soil  
fertility status documented in Table 1. The treatment effect was highly significant at the  
1% probability level (F = 14.34, p < 0.0001), confirming that the graded copper  
fertilization rates produced statistically significant differences in cane yield over and  
above the uniform NPK basal dose. The location × treatment interaction was marginally  
non-significant at the 5% probability level (F = 2.29, p = 0.0825), indicating that the  
broad direction of the cane yield response to Cu fertilization was consistent across the  
three contrasting sites, although location-specific divergences existed at the higher end of  
the Cu series.  
The simultaneous significance of the main treatment effect and broad consistency of the  
response across sites are particularly valuable for extension-scale interpretation. They  
imply that the optimum Cu rate identified through this trial can be recommended as a  
regional input for sugarcane growers across the Bahawalpur–Bahawalnagar tract without  
requiring detailed site-specific calibration based on soil EC, P, or K status. Comparable  
consistency of micronutrient response across contrasting soil fertility gradients has been  
reported for Zn in wheat and rice on Pakistani alkaline soils (Rashid and Ryan, 2004)  
and for Cu in cereals grown on calcareous soils (Rahman et al., 2022), supporting the  
generalizability of the present finding within similar agro-ecological zones.  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 14  
Table 3. Combined analysis of variance for cane yield of sugarcane across three locations  
and five copper fertilization levels.  
Source of variation  
DF Sum of squares Mean square F-value p-value  
Replications  
2
2
2
4
4,784,794  
1.21 × 10⁸  
5,228,410  
3.38 × 10⁸  
2,392,397  
1.21 × 10⁸  
2,614,205  
8.45 × 10⁷  
—
—
Locations (L)  
Error (a)  
46.36 0.0209*  
—
—
Treatments (T)  
14.34 <0.0001*  
*
L × T interaction  
Error (b)  
8
2.29  
5.41 × 10⁷  
1.35 × 10⁷  
0.0825ⁿˢ  
30  
5,892,974  
—
—
1.77 × 10⁸  
Total  
44  
Note: * = significant at 5% probability level; ** = significant at 1% probability level; ns  
= non-significant.  
3.3 Cane yield response to copper fertilization  
Across the pooled mean of the three locations, soil-applied copper sulphate pentahydrate  
produced a progressive improvement in cane yield as the Cu rate increased from 0 to 12  
kg ha⁻¹ (Figure 1). The unfertilized NPK-only control (T1) recorded the lowest pooled  
mean cane yield of 104,993.8 kg ha⁻¹, while application of 6, 8, 10, and 12 kg  
CuSO₄·5H₂O ha⁻¹ produced pooled mean yields of 108,139.5, 110,707.9, 112,358.0,  
and 112,843.2 kg ha⁻¹, respectively (Table 4). The yield response over the unfertilized  
control therefore rose from +3,145.7 kg ha⁻¹ (+3.0%) at T2 to +7,849.4 kg ha⁻¹  
(+7.5%) at T5, with the largest absolute yield gain recorded at the highest tested rate.  
The location-wise yield performance is presented in Figures 2, 3, and 4 for  
Haroonabad-1, Haroonabad-2, and Bahawalpur, respectively.  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 15  
This dose–response pattern is physiologically coherent and aligns with established  
understanding of copper nutrition in graminaceous crops. Adequate Cu supply enhances  
the activity of plastocyanin, an essential electron carrier on the donor side of  
photosystem I, and thereby supports photosynthetic carbon assimilation through the  
grand growth phase of the cane crop (Yruela, 2009; Printz et al., 2016). Cu is  
simultaneously a structural component of Cu/Zn superoxide dismutase, the principal  
cytosolic antioxidant enzyme protecting expanding tissues against reactive oxygen species  
generated under high-irradiance and salinity stress, conditions that prevail at all three of  
the present sites (Adrees et al., 2015). Equally important for cane in particular, Cu  
activates lignin biosynthetic enzymes including polyphenol oxidase and laccases, thereby  
strengthening stalk integrity and reducing yield losses through lodging during the late  
grand growth and maturation phases (Printz et al., 2016; Hunter et al., 2023). The  
magnitude of the yield gain recorded at the highest Cu rate; +7,849.4 kg ha⁻¹ over the  
unfertilized control on a pooled-mean basis, suggests that copper has been a latent but  
quantitatively important limitation on cane productivity at all three sites, and that NPK-  
only fertilization schedules currently in widespread use in the region significantly  
underexploit the yield potential of CPF-246 on alkaline-calcareous soils.  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 16  
Figure 1 | Performance of sugarcane crop at five different cupper fertilization at three  
different locations  
Table 4. Site-wise and pooled mean cane yield of sugarcane (cv. CPF-246) under five  
graded levels of copper fertilization across three locations of southern Punjab during  
2023–24.  
Treatment  
Haroonabad- Haroonabad- Bahawalpur  
Pooled  
Gain over  
T1 (%)  
mean (kg  
1 (kg ha⁻¹)  
2 (kg ha⁻¹)  
(kg ha⁻¹)  
ha⁻¹)  
T1 (0 kg)  
T2 (6 kg)  
T3 (8 kg)  
T4 (10 kg)  
103,629.6  
106,685.2  
109,703.7  
110,759.2  
106,629.6  
111,259.2  
114,481.5  
117,740.7  
104,722.2  
106,474.1  
107,938.5  
108,574.1  
104,993.8  
108,139.5  
110,707.9  
112,358.0  
—
+3.0  
+5.4  
+7.0  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 17  
Treatment  
T5 (12 kg)  
Haroonabad- Haroonabad- Bahawalpur  
Pooled  
Gain over  
T1 (%)  
mean (kg  
1 (kg ha⁻¹)  
2 (kg ha⁻¹)  
(kg ha⁻¹)  
ha⁻¹)  
113,481.5  
115,222.2  
109,825.9  
112,843.2  
+7.5  
3.4 Location-wise response and the location × treatment interaction  
When the data were disaggregated by location, two consistent and one site-specific  
pattern emerged (Figures 2–4 and Table 4). Haroonabad-2 recorded the highest mean  
cane yields across all treatments, ranging from 106,629.6 kg ha⁻¹ at T1 to a peak of  
117,740.7 kg ha⁻¹ at T4 (10 kg ha⁻¹), followed by a small decline to 115,222.2 kg ha⁻¹  
at T5 (12 kg ha⁻¹). Haroonabad-1 and Bahawalpur, by contrast, showed monotonically  
rising responses across the full Cu series, with yield at Haroonabad-1 climbing from  
103,629.6 kg ha⁻¹ (T1) to 113,481.5 kg ha⁻¹ (T5); a gain of +9,851.9 kg ha⁻¹, and at  
Bahawalpur from 104,722.2 kg ha⁻¹ (T1) to 109,825.9 kg ha⁻¹ (T5); a gain of  
+5,103.7 kg ha⁻¹. The absolute yield ranking across locations corresponds closely to the  
soil fertility status documented in Table 1: Haroonabad-2 carried the highest available P  
(10.9 ppm) and surface organic matter (0.71%), Haroonabad-1 was intermediate, and  
Bahawalpur carried the lowest available P (5.71 ppm) and K (90 ppm), explaining its  
lower baseline yield even under uniform NPK basal fertilization.  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 18  
Figure 2 | Yield performance of sugarcane crop at five different cupper fertilization  
levels at Haroonabad-1 location and difference from control  
Figure 3 | Yield performance of sugarcane crop at five different cupper fertilization  
levels at Haroonabad-2 location and difference from control  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 19  
Figure 2 | Yield performance of sugarcane crop at five different cupper fertilization  
levels at Bahawalpur location and difference from control  
The location × treatment interaction (F = 2.29, p = 0.0825) was marginally non-  
significant at the 5% probability level. The principal driver of this borderline interaction  
was the divergent yield trajectory at Haroonabad-2, where the response curve plateaued  
and slightly declined at T5, while the responses at the other two sites continued to rise  
across the full Cu series. The Haroonabad-2 yield dip at the highest Cu rate may reflect  
the comparatively better baseline P and OM status at that site, which would have  
permitted higher tissue Cu loading and brought the crop closer to the sufficiency  
threshold beyond which further Cu supply ceases to be agronomically beneficial.  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 20  
Antagonistic interactions of Cu with Zn and Fe uptake at high tissue Cu concentrations  
are well documented in graminaceous crops (Adrees et al., 2015; Rahman et al., 2022)  
and provide a plausible mechanism for the modest yield depression observed at T5 on  
the more fertile of the three sites. From an extension standpoint, however, the formal  
non-significance of the interaction term, together with the consistency of the broad  
treatment ranking across all three sites, supports the use of a regional Cu  
recommendation rather than separate site-specific calibrations.  
3.5 Identifying the recommended Cu rate  
Taking the pooled and location-wise yield responses together, the T5 treatment (12 kg  
CuSO₄·5H₂O ha⁻¹) produced the largest absolute cane yield response over the  
unfertilized NPK-only control on a pooled-mean basis and is therefore identified as the  
recommended Cu rate for spring-planted CPF-246 sugarcane on the alkaline-calcareous  
soils of southern Punjab. At this rate, the pooled mean cane yield reached 112,843.2 kg  
ha⁻¹, representing an absolute gain of +7,849.4 kg ha⁻¹ (+7.5%) over the NPK-only  
control. The yield gain at T5 was substantial and consistent across all three sites, ranging  
from +5,103.7 kg ha⁻¹ at Bahawalpur to +9,851.9 kg ha⁻¹ at Haroonabad-1, indicating  
that 12 kg CuSO₄·5H₂O ha⁻¹ delivered the largest reproducible cane yield response and  
represented the upper end of the agronomic response curve within the rates tested. Given  
the comparatively low cost of CuSO₄·5H₂O against the substantial absolute yield  
response, this rate emerges as both an agronomically and economically defensible  
recommendation. Final cane yield in sugarcane is jointly determined by stand density  
and individual cane biomass (Bull and Glasziou, 1975), and the consistent yield gains  
observed under T5 indicate that copper supply at this level adequately addresses both of  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 21  
these yield components on alkaline-calcareous soils where Cu bioavailability is otherwise  
constrained by carbonate fixation.  
It should be noted that the pooled-mean yield advantage from T4 to T5 was modest  
(+485.2 kg ha⁻¹; +0.43%), and that the response at Haroonabad-2 specifically peaked  
at T4 (10 kg ha⁻¹) rather than at T5. The flattening of the response curve at the more  
fertile Haroonabad-2 site, and the small but consistent additional gains observed at the  
two lower-fertility sites, together suggest that the true sufficiency threshold for soil-  
applied Cu under these alkaline-calcareous conditions lies at or near 12 kg  
CuSO₄·5H₂O ha⁻¹. The marginal yield improvement at T5 also signals that Cu supply  
beyond this rate is unlikely to deliver additional agronomic benefit and may begin to  
introduce risks of induced Zn or Fe deficiency, particularly on soils where these  
micronutrients are already marginal (Rahman et al., 2022). Validation of the present  
recommendation under ratoon-crop conditions, autumn-planted cane, and a broader soil  
fertility gradient would further strengthen its applicability across the wider sugarcane  
cropping systems of southern Punjab.  
4. Conclusion  
The present multi-location field investigation demonstrated that soil-applied copper  
sulphate pentahydrate, broadcast at planting alongside a uniform NPK basal dose,  
produced a statistically significant and agronomically meaningful improvement in cane  
yield of spring-planted sugarcane (cv. CPF-246) under the alkaline-calcareous, canal-  
irrigated conditions of southern Punjab. Pooled mean cane yield rose progressively from  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 22  
104,993.8 kg ha⁻¹ in the unfertilized control to 112,843.2 kg ha⁻¹ at the highest tested  
dose, representing an absolute yield gain of +7,849.4 kg ha⁻¹ (+7.5%) at 12 kg  
CuSO₄·5H₂O ha⁻¹. The yield response was monotonic and still rising at Haroonabad-1  
and Bahawalpur, while at the more fertile Haroonabad-2 site the response peaked at 10  
kg ha⁻¹ and declined slightly at 12 kg ha⁻¹, generating a marginally non-significant  
location × treatment interaction (p = 0.0825). On the strength of the largest absolute  
yield response over the unfertilized control across the pooled and site-level analyses, the  
inclusion of 12 kg CuSO₄·5H₂O ha⁻¹ as a basal soil-broadcast input is recommended as  
a regional fertilizer practice for sugarcane growers across the Bahawalpur–Bahawalnagar  
tract, complementing rather than replacing the existing NPK schedule. The findings  
further argue for a broader re-examination of copper, and of micronutrient nutrition in  
general, in the fertilizer recommendations issued for sugarcane on alkaline soils of  
Pakistan, where decades of NPK-centric fertilization have left a latent but evidently  
corrigible Cu limitation on productivity. Future work should extend the present trial to  
ratoon-crop performance, to autumn-planted cane, and to wider soil fertility gradients,  
and should additionally evaluate the response of juice quality parameters such as Brix,  
sucrose percentage, and commercial cane sugar to graded Cu fertilization, so that a fully  
integrated micronutrient recommendation for sugarcane in southern Punjab can be  
developed.  
Author Contributions  
MJQ; Conceptualization and Execution, Supervision, Planning, Writing – original draft;  
MRF, SJ; Experimental layout, Field execution and crop management at Haroonabad-1  
and Haroonabad-2 sites; MBL, AR; Field execution and crop management at  
Bahawalpur site, Treatment application; AAS, BB; Data analysis, Statistical computation,  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 23  
Data visualization; MA, TA; Soil sampling, Soil chemical analysis, Soil fertility  
characterization; SH, MIM, MAU; Yield data recording, Data compilation; MJQ, AAS,  
MRF; Writing – review & editing.  
Conflict of interest  
The authors declared absence of conflict of interest.  
References  
1. Government of Pakistan (GoP) (2024). Pakistan Economic Survey 2023–24,  
Chapter 2: Agriculture. Finance Division, Economic Adviser's Wing, Islamabad.  
2. Afghan, S., Khan, M. E., Verma, K. K., & Nikpay, A. (2024). Economic importance  
and yield potential of sugarcane in Pakistan. In Sugarcane cultivation and  
management (pp. 263-291). Apple Academic Press.  
3. Shahzad, A.N., Qureshi, M.K., Wakeel, A., Misselbrook, T. (2019). Crop  
production in Pakistan and low nitrogen use efficiencies. Nature Sustainability,  
2(12): 1106–1114.  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 24  
4. Majeed, A., Rashid, I., Niaz, A., Ditta, A., Sameen, A., Al-Huqail, A.A., Siddiqui,  
M.H. (2022). Balanced use of Zn, Cu, Fe, and B improves the yield and sucrose  
contents of sugarcane juice cultivated in sandy clay loam soil. Agronomy, 12(3): 696.  
5. Manzoor, M., Khan, M.Z., Ahmad, S., Alqahtani, M.D., Shabaan, M., Sarwar, S.,  
Hameed, M.A., Zulfiqar, U., Hussain, S., Ali, M.F., Ahmad, M., Haider, F.U.  
(2023). Optimizing sugarcane growth, yield, and quality in different ecological zones  
and irrigation sources amidst environmental stressors. Plants, 12(20): 3526.  
https://doi.org/10.3390/plants12203526  
6. Rashid, A., Ryan, J. (2004). Micronutrient constraints to crop production in soils  
with Mediterranean-type climates: a review. Journal of Plant Nutrition, 27(6): 959–  
975.  
7. Imran, M., Rehim, A., Sarwar, N., Hussain, S. (2020). Zinc bioavailability in maize  
grains in response to phosphorus–zinc interaction. Journal of Plant Nutrition and  
Soil Science, 183(3): 305–311.  
8. Zia, M.H., Ahmad, R., Khaliq, I., Ahmad, A., Irshad, M. (2007). Micronutrients  
status and management in orchards soils: applied aspects. Soil and Environment,  
26(1): 13–22.  
9. Yruela, I. (2009). Copper in plants: acquisition, transport and interactions.  
Functional Plant Biology, 36(5): 409–430.  
10.Printz, B., Lutts, S., Hausman, J.F., Sergeant, K. (2016). Copper trafficking in plants  
and its implication on cell wall dynamics. Frontiers in Plant Science, 7: 601.  
11.Adrees, M., Ali, S., Rizwan, M., Ibrahim, M., Abbas, F., Farid, M., Zia-ur-Rehman,  
M., Irshad, M.K., Bharwana, S.A. (2015). The effect of excess copper on growth and  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 25  
physiology of important food crops: a review. Environmental Science and Pollution  
Research, 22(11): 8148–8162.  
12.Hunter, A.S., Bagniewska-Zadworna, A., Sokolowska, K., Brodribb, T.J. (2023). The  
effects of copper deficiency on lignification, xylem vessel structure, and hydraulic  
traits in hybrid poplar. Physiologia Plantarum, 175(5): e14006.  
13.McCray, J.M., Rice, R.W., Ezenwa, I.V., Lang, T.A., Baucum, L. (2010). Sugarcane  
plant nutrient diagnosis. University of Florida IFAS Extension, Document SS-AGR-  
128. University of Florida, Gainesville, FL.  
14.Naga Madhuri, K.V., Sarala, N.V., Hemanth Kumar, M., Subba Rao, M., Giridhar,  
V. (2013). Influence of micronutrients on yield and quality of sugarcane. Sugar  
Tech, 15(2): 187–191. https://doi.org/10.1007/s12355-012-0196-3  
15.Ryan, J., Estefan, G., Rashid, A. (2001). Soil and Plant Analysis Laboratory Manual,  
2nd ed. International Center for Agricultural Research in the Dry Areas (ICARDA),  
Aleppo, Syria.  
16.Estefan, G., Sommer, R., Ryan, J. (2013). Methods of Soil, Plant, and Water  
Analysis: A Manual for the West Asia and North Africa Region, 3rd ed.  
International Center for Agricultural Research in the Dry Areas (ICARDA), Beirut,  
Lebanon.  
17.Steel, R.G.D., Torrie, J.H., Dickey, D.A. (1997). Principles and Procedures of  
Statistics: A Biometrical Approach, 3rd ed. McGraw-Hill Book Co., New York.  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03  
G. 2112  
Page 26  
18.Rahman, N., Peak, D., Schoenau, J. (2022). Antagonistic effect of copper and zinc  
in fertilization of spring wheat under low soil phosphorus conditions. Canadian  
Journal of Soil Science, 102(3): 797–809. https://doi.org/10.1139/cjss-2021-  
0189  
19.Bull, T.A., Glasziou, K.T. (1975). Sugarcane. In: L.T. Evans (ed.), Crop Physiology:  
Some Case Histories. Cambridge University Press, Cambridge, UK, pp. 51–72.  
20.FAO (2023). World Food and Agriculture – Statistical Yearbook 2023. Food and  
Agriculture Organization of the United Nations, Rome.  
21.FAOSTAT (2023). Crops and livestock products database. Food and Agriculture  
Organization of the United Nations. Available at: https://www.fao.org/faostat  
(accessed 2024).  
GRJNST, Volume: 04 - Issue 4 (2026) / ISSN P: 2790-7643  
Article ID: 2112  
https://doi.org/10.53762/grjnst.04.04.03