Multi-locational study on plant growth regulators to minimize pre-mature fruit drop and maximize postharvest quality of apples

Abstract

Plant growth regulators (PGR’s) are considered potential substances which lessen the abiotic stresses and help in delaying the pre-mature fruit drop (PFD). Therefore, a two-year (2021 and 2022) multi-locational study was designed to assess the effects of three different PGR’s (NAA, 2, 4-D, GA₃) on PFD and postharvest quality of selected apple varieties (‘Kashmir Amri’, ‘Red Delicious’, ‘Star Crimson’) grown at three different locations (Bagh, Dheerkot, Rawalakot) of Azad Jammu and Kashmir (AJK). All PGR’s were applied through foliar spray using a low pressure hand sprayer before the flowering period at the same concentration (25 ppm), while apple trees without any treatment application served as control. Apple trees sprayed with 25 ppm GA₃ showed 44.56% less PFD at Dheerkot location as compared to control. Moreover, 25 ppm GA₃ treated fruits showed better fruit size, fruit weight, and overall yield as well. In terms of postharvest quality parameters, a significantly high amount of strife index (5.60%), titratable acidity (2.58%), total soluble solids (13.81%), crude fiber (1.44%), total ash content (0.51%), total phenolics (136.3 mg GAE/100g FW), total flavonoids (14.83 mg/100g FW) and antioxidant activity (97.0 µg/100g FW) was found in apple trees sprayed with 25 ppm GA₃ as compared with other treatments. Among apple varieties, ‘Star Crimson’ while among locations, Dheerkot showed promising results. Thus, the overall results of this study revealed that PFD could be potentially minimized and postharvest quality could be enhanced with 25 ppm GA₃ spray, while Dheerkot is the most suitable location for growing apples.

Keywords:

• Naphthalene acetic acid Gibberellic acid 2
• 4-Dichlorophenoxyacetic acid Fruit drop percentage Postharvest quality Antioxidant activity

REVIEWING EDITOR:

• Manuel Tejada Moral, University of Seville, Spain

SUBJECTS:

• Agriculture and Natural Resources; R&D; Agriculture Technology

1. Introduction

Rapid changes in climatic conditions impose various stresses on plants, directly impacting their growth patterns and nutritional properties (Abdel-Sattar et al., 2023). Therefore, maintaining a harmonious balance between the climatic requirements of plants and the prevailing climatic conditions in a region is crucial. Abiotic factors such as extremely cold conditions during flowering and fruit set, hailstorms, and hot and humid weather affect almost all plant functions and maturity indices of fruits and vegetables (Malik & Deveshwar, 2017). If these conditions prevail for longer periods, serious losses occur in fruit production due to increased pre-mature fruit drop (PFD; Bolat et al., 2014).

Apple (Malus domestica Borkh.) is one of the most important temperate fruit crops with significant economic and nutritional value. Asia, Central Asia, Himalayan regions of India, Pakistan and Western China are believed as the center of origin for apples (Muzher et al., 2007). This crop covered approximately five million acres of land around the world with the production of 93.11 million metric tonnes of apples in 2021 (Abdel-Sattar & Kotb, 2021). China ranked first in apple production with a total volume of 46 million metric tonnes during 2017, followed by USA (Statistica, 2018).

In Pakistan apple is successfully cultivated in temperate hilly tracts of Quetta, Zairat, Pishin, Mustang, Kalat, Murree hills of Punjab, Chitral, Swat, Manshera, Kalam, Gilgit, Sakrdu, Chilas, Hunza and Kashmir. However, apple production in Pakistan is quite low compared to other countries of the world. Therefore, Pakistan stands at the 23rd position among apple producing countries in the world. The cultivated area of apple orchards was around 88.80 thousand hectares, having an average fruit production of 482 thousand tonnes during 2018–2019 (Government of Pakistan (GOP), 2018).

Among other factors, one of the main factors behind this low apple production is considered PFD, which causes almost 50% of yield loss (Arseneault & Cline, 2018). Due to different abiotic stresses, PFD in apples is due to high respiration rate and ethylene production which causes cell separation in the pedicle and consequently results in fruit shed.

Plant growth regulators (PGR’s) are substances which lessen abiotic stresses by altering plant physiology and reduction in ethylene production, thus resulting in a delay in PFD (Arseneault & Cline, 2018; Öztürk et al., 2015). The most commonly used PGR is 1-naphthalene acetic acid (NAA), which helps in down- regulating the function of genes which are responsible for cell separation and promote PFD (Öztürk et al., 2012). NAA particularly is responsible for the reduction of the sensitivity of the abscission zone to ethylene production which results in the prevention of PFD (Özkan et al., 2016). 2, 4-Dichlorophenoxyacetic acid (2,4-D) is another commonly used auxin which reduces the fruit drop by preventing the synthesis of hydrolytic enzymes such as cellulose which is responsible for the decomposition of cell wall (Modise et al., 2009). Among PGR’s, gibberellins (GAs) are a category of tetracyclic diterpenoid hormones in higher plants regulating a wide range of developmental processes (Shu et al., 2016; Liu et al., 2022). An exogenous GA₃ treatment not only reduced the ethylene production but also depressed the ripening of various climacteric fruits, such as bananas, persimmons, and tomatoes (Li et al., 2019; Chen et al., 2016).

Azad Jammu and Kashmir (AJK) is blessed with suitable climatic conditions for growing apples. Currently, apple is being cultivated as a major fruit crop in five Districts of AJK including Poonch, Bagh, Sudhnoti, Muzaffarabad, and Neelum. Total area under cultivation in AJK is still unknown as there is mostly scattered plantation of apples in this region. However, in a recent report published by Department of Agriculture, AJK, the number of apple trees were noted around 3.0 million with a production volume of 60 thousand tonnes (Javeid & Nawab, 2021). A number of varieties including ‘Kashmir Amri’, ‘Star Crimson’, ‘Red Chief’ and ‘Red Delicious’ can be found in these Districts (Ahmed & Raza, 2005). However, due to multiple factors including mechanical damage and other abiotic factors such as hail and wind storms before harvest, fruit yield has been observed low during the last few years. These factors resulted in a severe PFD of apples in this area. In some varieties, this drop was up to 20%, while in some other varieties, it was as high as 50%. In the recent past, this PFD of apples in AJK has resulted in severe economic losses to the local growers.

Therefore, there is an enormous need and scope to work on this local challenge in this area. So far, no studies have been conducted to determine the actual cause of this problem and its management in AJK. Thus, it is hypothesized that use of PGR’s may reduce the PFD and improve postharvest quality of selected apple varieties grown at different locations. In this regard, the present study was designed to explore the influence of different PGR’s (NAA, 2, 4-D, and GA₃) on PFD and postharvest quality of selected apple varieties (‘Kashmir Amri’, ‘Red Delicious’ and ‘Star Crimson’) grown at three different locations (Bagh, Dheerkot and Rawalakot) of AJK.

2. Materials and methods

2.1. Field site description and study details

This study was performed at three different locations [Bagh (Latitude: 33.585880˚N; Longitude: 73.465880˚E; Elevation: 1086 m), Dheerkot (Latitude: 34.0389733˚N; Longitude: 73.5770801˚E; Elevation 1673 m) and Rawalakot (Latitude: 33.8583729˚N; Longitude: 73.7654367˚E; Elevation 1671 m)] of Azad Jammu and Kashmir in three different apple varieties (‘Kashmir Amri’, ‘Red Delicious’, ‘Star Crimson’). The study was carried out during consecutive seasons of year 2021 and year 2022. Apple trees trained using modified-central leader system and grafted on MM111 rootstock were selected based on their health, uniformity in vigour, and the age (10–12 years). These trees were spaced 3 × 4 m apart and grown in sandy loam soils. All management practices remained the same in these areas. Fertilizers (2.5 kg Potash, 1.5 kg Urea, 750 g DAP) were applied to each tree. Urea was applied in three split doses before flowering, at the marble stage and before harvest. Weeding and irrigation were also practiced as per need (Irfan et al., 2020). All PGR treatments, i.e. NAA (25 ppm), GA₃ (25 ppm), and 2, 4-D (25 ppm) were applied through foliar spray using a low-pressure hand sprayer (GT 1, Sky Seeds Pvt. Ltd.) before the flowering period. Apple trees without any treatment application served as control. Each treatment consisted of six trees; three trees were used to monitor PFD and the other three were used to sample fruits for yield and postharvest quality analysis. Five branches (four from each side and one from the middle) of each tree were tagged in order to study the effects of PGRs. Apple fruits were hand harvested (100 fruits per tree) on 27 August 2021 and 22 August 2022 from three different locations (Rawalakot, Bagh, and Dheerkot). Fruits were transported immediately (25 ± 3 °C, 60%–70% RH) using University van to the Laboratory of Department of Horticulture, Faculty of Agriculture, University of Poonch Rawalakot for further analysis.

2.2. Fruit productivity parameters

Data collection for fruit productivity was done by several parameters. Number of flowers per branch from five branches of each apple tree were counted and the average was taken. Fruit set percentage was calculated by dividing the number of fruits per panicle of perfect flowers. Fruit drop (%) was calculated using the following formula. $$\text{Fruit \hspace{0.33em}drop}\hspace{0.17em}(\text{\%})\text{\hspace{0.33em}=\hspace{0.33em}}\hspace{0.17em}\frac{\begin{array}{l}\text{No. \hspace{0.33em}of \hspace{0.33em}fruit \hspace{0.33em}drop \hspace{0.33em}from \hspace{0.33em}}28\text{\hspace{0.33em}days\hspace{0.33em}}\hspace{0.17em}\\ \text{before\hspace{0.33em} anticipated\hspace{0.33em} harvest}\hspace{0.17em}\text{\hspace{0.33em}until\hspace{0.33em} harvest}\end{array}}{\text{Total \hspace{0.33em}number\hspace{0.33em} of\hspace{0.33em} fruits}}\times 100$$

Fruit size (cm²) was recorded by measuring the length and width of harvested fruits with the help of Vernier caliper (Model: Insize SR44) and their average was taken. To measure fruit weight (g), 25 fruits were randomly selected from each replication in each treatment and weighed with the help of a weighing balance (Model: Shimadzu A ×20). Yield per tree (kg) was determined by measuring the weight of all fruits from each tree with the help of weighing balance. Fruit firmness was measured using a hand-held penetrometer (Model: GY. 4), having 1.0-inch diameter probe. Fruit juice (%) was determined by extracting the juice from a unit weight of apple fruit sample. Fruit juice (%) was calculated by using the following formula. $$\text{Fruit juice}\hspace{0.17em}\left(\%\right)\hspace{0.17em}=\hspace{0.17em}\frac{\text{Juice weight}\hspace{0.17em}+\hspace{0.17em}\text{Beaker}\hspace{0.17em}-\hspace{0.17em}\text{Beaker weight}}{\text{Weight of}\hspace{0.17em}\text{apples}}\times 100$$

2.3. Fruit quality indices

Strife index (%) is considered as a maturity index and was calculated using the following formula. $$\text{Strife index}\mathrm{(}\text{\%}\mathrm{)}\text{=}\frac{\text{Fruit firmness}}{\text{Brix}20\mathrm{˚}\mathrm{x}\text{Starch iodine index}}\mathrm{x}100$$

Juice was extracted from apple fruits for measuring pH (AOAC, 1990). pH meter (Model: WTW 82362, Inolab, Germany) was used for this purpose. Titratable acidity in terms of malic acid equivalents in apple samples was estimated (AOAC, 1990). For this purpose, the pulp of apple fruits (5 g) was taken to homogenize with purified water (20 mL). After homogenization, the mixture was filtered, and the pure extract was obtained. 5 mL extract was used from each sample for titration with 0.1 N NaOH solution, and phenolphthalein was used as an indicator and expressed in percentage of malic acid. Hand-held refractometer (ATAGO Co. Ltd., Japan) was used to measure total soluble solids. Readings were taken at room temperature and expressed in percentage (AOAC, 1990). To measure the crude fiber apple fruit sample (5 g) was dried in a hot oven to obtain a constant weight (AOAC, 1990). After that, the sample was digested using 1.25% sulphuric acid and 1.25% sodium hydroxide, respectively. Washing was done for the digested sample using purified water and then kept in a furnace at 500 or 550 °C to obtain white ash. Following formula was used to calculate the crude fiber. $$\text{Crude fiber}\hspace{0.17em}\text{(}\%\text{)}\hspace{0.17em}=\frac{\left(c-b\right)-\text{(b}-\text{d)}}{\text{(a)}}\times 100$$ a = Sample weight b = Crucible weight

c = Sample weight before ignition d = Sample weight after ignition

Muffle furnace (Model: SX-2-5-10) was used at 600 °C for 3 hours to measure the ash content in apple fruit samples. Total ash content were calculated by taking the difference in fresh weight of samples and dried weight of samples and expressed in percentage (AOAC, 1998).

2.4. Health related compounds

2,6-Dichlorophenol indophenol dye was used to measure vitamin C in apple fruit samples (AOAC, 1990). Mixing of apple fruit extract (100 mL) and 4% meta-phosphoric acid solution (5 mL) was done. Later on the titration was done using dye until a light pink colour was appeared. To measure total phenolics in apple fruit samples, a standard curve prepared using gallic acid equivalent (GAE) was used, and the results were expressed as mg GAE/100g fruit weight (FW) (Maqbool et al., 2019). For measuring total flavonoids, mixing of 0.5 mg apple fruit pulp and 1.5 mL of methanolic AlCl₃.6H₂O was done in sealed tubes for 15 min and kept in a dark room (Ali et al., 2014). After mixing, the absorbance was taken using spectrophotometer (Model: UV 4000, ORI, Reinbeker, Hamburg, Germany) at 430 nm and the results were shown as mg/100g FW (Zahid et al., 2014). Total antioxidants were calculated by using Ferric Reducing Antioxidant Power (FRAP) assay (Zahid et al., 2022). For this analysis, 40 µL of apple fruit extract was mixed with 3 mL of FRAP reagent and incubated for 4 min in the dark. Absorbance (593 nm) was taken and the results were shown as the activity of FeSO₄ as µg/100g FW.

2.5. Statistical analyses

The experiment was laid out in a Randomized Complete Block Design (RCBD) with three-factor factorial (variety, locations, and treatments) having three replicates. ANOVA using Statistical Software (Statistix 8.1) was taken using the collected data and HSD Tukey’s test at p ˂ .05 was used to compare means. The results of ANOVA summary for all parameters are presented in Table 1. To check the strength of association for various variables, Pearson correlation was done using corrplot function, while for drawing the biplot between PC1 and PC2, fviz_pca_biplot was used and for this analysis R Statistical Software (version 4.1.1) was used. Repetition of all trials was done to confirm the reproducibility of results. However, the data of repeated trials were combined for analysis, as the key results of a single analysis revealed at par results across the trials.

Table 1. ANOVA summary for fruit productivity parameters, fruit quality indices and health-related compounds.

Factors	No. of flowers per branch		Fruit set (%)		Fruit drop (%)		Fruit size (cm^2)
	p-value	F	p-value	F	p-value	F	p-value	F
Loc x Treat	<.05	17.68	<.05	53.91	<.05	53.91	.2866	1.26
Loc x Var	<.05	3.83	<.05	0.47	<.05	0.47	<.05	5.57
Treat x Var	<.05	2.27	<.05	2.05	<.05	2.05	<.01	6.87
Loc x Treat x Var	<.01	2.37	<.01	2.10	<.05	2.10	<.05	1.94
CV	4.77		2.16		2.79		4.03
Factors	Fruit weight (g)		Yield per tree (kg)		Fruit firmness (kg/cm^2)		Fruit juice (%)
	p-value	F	p-value	F	p-value	F	p-value	F
Loc × Treat	<.01	3.20	<.01	44.50	<.01	25.76	<.05	3.30
Loc × Var	<.05	2.66	.9875	0.08	.3721	1.08	<.05	3.31
Treat × Var	<.01	4.20	<.01	37.85	<.01	26.76	<.05	45.06
Loc × Treat × Var	<.05	1.93	<.01	3.32	<.01	3.09	<.05	2.70
CV	9.51		1.28		4.72		3.47
Factors	Strife index (%)		pH		Titratable acidity (%)		Total soluble solids (%)
	p-value	F	p-value	F	p-value	F	p-value	F
Loc × Treat	<.05	2.68	<.05	2.80	<.01	5.05	.0560	2.22
Loc × Var	.1210	1.89	<.05	31.19	.3817	1.06	<.01	5.02
Treat × Var	<.01	26.28	<.05	4.62	<.01	6.44	<.01	11.29
Loc × Treat × Var	<.01	3.48	<.05	2.19	<.05	1.98	<.05	2.04
CV	6.36		3.58		10.78		8.78
Factors	Crude fiber (%)				Total ash content (%)
	p-value		F		p-value		F
Loc × Treat	<.01		5.88		<.01		5.41
Loc × Var	.1631		1.69		.08		2.17
Treat × Var	<.05		2.98		<.01		5.44
Loc × Treat × Var	<.01		3.68		<.05		1.85
CV	5.61				9.18
Factors	Vitamin C (mg/100g)		Total phenolics (mg gallic acid/100g FW)		Total flavonoids (mg/100g FW)		Antioxidant activity (µg/100g FW)
	p-value	F	p-value	F	p-value	F	p-value	F
Loc × Treat	.2223	1.41	<.01	9.32	<.01	5.16	<.05	3.28
Loc × Var	<.05	2.63	<.01	4.48	.0772	2.21	.2898	1.27
Treat × Var	<.01	15.01	<.01	16.22	<.01	5.13	<.01	19.02
Loc × Treat × Var	<.05	2.60	<.05	2.48	<.05	2.28	<.01	3.40
CV	6.26		6.33		5.53		7.61

Loc × Treat: Locations × Treatments; Loc × Var: Locations × Varieties; Treat × Var: Treatments × Varieities; Loc × Treat × Var: Locations × Treatments × Varieties; CV:.

3. Results

3.1. Fruit productivity parameters

All fruit productivity parameters showed significant (p ˂ .05) differences among all PGR treatments, locations and apple varieties (Table 1). Number of flowers per branch were recorded maximum in ‘Red Delicious’ apples (195.3) which were sprayed with GA₃ and grown at Dheerkot location. These results were at par with ‘Star Crimson’ apples (188.0) which were treated with GA₃ and grown at the same location. Whereas, control trees showed the minimum number of flowers per branch in all three apple varieties and at all locations (Figure 1(a)). Fruit set (%) was noted the highest in ‘Star Crimson’ apples (78.3%) which were sprayed with GA₃ and grown at Dheerkot location. While, control trees showed the lowest fruit set (%) in all three apple varieties and at all locations (Figure 1(b)). Fruit drop (%) was recorded maximum in untreated control trees of ‘Kashmir Amri’ apples (75.33%) which were grown at Rawalakot location. Whereas, the minimum fruit drop (%) was observed in ‘Star Crimson’ apples (21.0%) which were treated with GA₃ at Dheerkot location (Figure 1(c)). Fruit size (cm²) was observed maximum in ‘Star Crimson’ apples (99 cm²) which were treated with GA₃ at Dheerkot location. Whereas, untreated control trees of ‘Kashmir Amri’ apples showed the minimum fruit size (32.3 cm²) at Rawalakot location (Figure 2(a)). ‘Star Crimson’ apples sprayed with GA₃ and grown at Dheerkot and Bagh locations attained the maximum fruit weight (213.6 g), which was at par with ‘Red Delicious’ apples (207.0 g) and grown at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed minimum fruit weight at all locations (Figure 2(b)). Yield per tree (kg) was found maximum in ‘Star Crimson’ apples (1309.0 kg) treated with GA₃ and grown at Dheerkot location, which was at par with ‘Red Delicious’ apples (1285.3 kg) sprayed with the same treatment and grown at the same location. Whereas, control trees of ‘Kashmir Amri’ apples showed minimum yield at all locations (Figure 2(c)). Fruit firmness (kg/cm²) was noted maximum in ‘Star Crimson’ apples (12.76 kg/cm²) which were treated with GA₃ and grown at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum fruit firmness at all locations (Figure 3(a)). Fruit juice (%) was observed maximum in ‘Star Crimson’ apples (89.6%) treated with GA₃ at Dheerkot location, which was at par with ‘Red Delicious’ apples (88.3%) sprayed with the same treatment and grown at the same location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum fruit juice at all locations (Figure 3(b)).

Figure 1. Effect of different plant growth regulators on three apple varieties (‘Kashmir Amri’, ‘Red Delicious’, ‘Star Crimson’) grown at three different locations (Bagh, Dheerkot, Rawalakot) of Azad Jammu and Kashmir on (a) Number of flowers per branch (b) Fruit set (%) (c) Fruit drop (%). Treatments [1: Control; 2: NAA (25 ppm); 3: GA₃ (25 ppm); 4: 2, 4-D (25 ppm)].

Figure 2. Effect of different plant growth regulators on three apple varieties (‘Kashmir Amri’, ‘Red Delicious’, ‘Star Crimson’) grown at three different locations (Bagh, Dheerkot, Rawalakot) of Azad Jammu and Kashmir on (a) Fruit size (cm²) (b) Fruit weight (g) (c) Yield per tree (kg). Treatments [1: Control; 2: NAA (25 ppm); 3: GA₃ (25 ppm); 4: 2, 4-D (25 ppm)].

Figure 3. Effect of different plant growth regulators on three apple varieties (‘Kashmir Amri’, ‘Red Delicious’, ‘Star Crimson’) grown at three different locations (Bagh, Dheerkot, Rawalakot) of Azad Jammu and Kashmir on (a) Fruit firmness (kg/cm²) (b) Fruit juice (%). Treatments [1: Control; 2: NAA (25 ppm); 3: GA₃ (25 ppm); 4: 2, 4-D (25 ppm)].

3.2. Fruit quality indices

All fruit quality indices showed significant (p ˂ .05) differences among all PGR treatments, locations and apple varieties (Table 1). Strife index (%) was observed maximum in ‘Star Crimson’ apples (5.60%) treated with GA₃ and grown at Dheerkot location, which was at par with ‘Red Delicious’ apples (5.10%) treated with GA₃ at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum strife index at all locations (Figure 4(a)). pH was observed maximum in ‘Star Crimson’ apples (4.10) which were treated with GA₃ and grown at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum pH at all locations (Figure 4(b)). Titratable acidity (%) was noted the highest in ‘Star Crimson’ apples (2.58%) treated with GA₃ and grown at Dheerkot location, which was at par with ‘Red Delicious’ apples (2.44%) treated with GA₃ at Dheerkot location. Whereas, control trees of Kahmir Amri apples showed the minimum titratable acidity at all locations (Figure 4(c)). Total soluble solids (%) were found maximum in ‘Star Crimson’ apples (13.81%) which were treated with GA₃ and grown at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum total soluble solids at all locations (Figure 4(d)). Crude fiber (%) was observed maximum in ‘Star Crimson’ apples (1.44%) which were treated with GA₃ and grown at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum crude fiber (%) at all locations (Figure 4(e)). Total ash content (%) were recorded maximum in ‘Star Crimson’ apples (0.51%) treated with GA₃ and grown at Dheerkot location, which were at par with ‘Red Delicious’ apple variety (0.50%) treated with GA₃ at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed minimum total ash content (%) at all locations (Figure 4(f)).

Figure 4. Effect of different plant growth regulators on three apple varieties (‘Kashmir Amri’, ‘Red Delicious’, ‘Star Crimson’) grown at three different locations (Bagh, Dheerkot, Rawalakot) of Azad Jammu and Kashmir on (a) Strife index (%) (b) pH (c) Titratable acidity (%) (d) Total soluble solids (%) (e) Crude fiber (%) (f) Total ash content (%). Treatments [1: Control; 2: NAA (25 ppm); 3: GA₃ (25 ppm); 4: 2, 4-D (25 ppm)].

3.3. Health related compounds

All health-related parameters showed significant (p ˂ .05) differences among all PGR treatments, locations and apple varieties (Table 1). Vitamin C (mg/100g) was observed maximum in ‘Star Crimson’ apples (9.30 mg/100g) which were treated with GA₃ and grown at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum vitamin C at all locations (Figure 5(a)). Total phenolics (mg gallic acid/100g FW) were recorded maximum in ‘Star Crimson’ apples (136.3 mg gallic acid/100g FW) which were treated with GA₃ and grown at Dheerkot location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum total phenolics at all locations (Figure 5(b)). Total flavonoids (mg/100g FW) were noted maximum in ‘Star Crimson’ apples (14.83 mg/100g FW) treated with GA₃ and grown at Dheerkot location, which were at par with ‘Red Delicious’ apples (13.40 mg/100g FW) sprayed with the same treatment and grown at the same location. Whereas, control trees of ‘Kashmir Amri’ apples showed the minimum total flavonoids at all locations (Figure 5(c)). Antioxidant activity (µg/100g FW) was found the highest in ‘Star Crimson’ apples (97.0 µg/100g FW) treated with GA₃ and grown at Dheerkot location, which was at par with ‘Red Delicious’ apples (92.6 µg/100g FW) sprayed with same treatment and grown at same location. Whereas, control trees of ‘Kashmir Amri’ apples showed the lowest antioxidant activity at all locations (Figure 5(d)).

Figure 5. Effect of different plant growth regulators on three apple varieties (‘Kashmir Amri’, ‘Red Delicious’, ‘Star Crimson’) grown at three different locations (Bagh, Dheerkot, Rawalakot) of Azad Jammu and Kashmir on (a) Vitamin C (mg/100g) (b) Total phenolics (mg gallic acid/100g FW) (c) Total flavonoids (mg/100g FW) (d) Antioxidant activity (µg/100g FW). Treatments [1: Control; 2: NAA (25 ppm); 3: GA₃ (25 ppm); 4: 2, 4-D (25 ppm)].

3.4. Principal component analysis of fruit productivity, fruit quality indices, and health- related compounds

Principal component analysis was carried out among different traits under observation to investigate the association among different traits of apple varieties. Biplot analysis between Dim 1 and Dim 2 explained 79% and 6.3% variability, respectively, and demonstrated that antioxidant activity, total flavonoids and vitamin C were clustered closer to total phenolics and fruit firmness, while opposite to fruit drop percentage (Figure 6).

Figure 6. Biplot of combined principal component analysis for fruit productivity parameters, fruit quality indices and health related compounds. Flo: Number of flowers per branch; Set: Fruit set; Dro: Fruit drop; Siz: Fruit size; Wei: Fruit weight; Yie: Yield per tree; Fir: Fruit firmness; Jui: Fruit juice; Stri: Strife index; TAA: Titratable acidity; TSS: Total soluble solids; Fib: Crude fiber; Ash: Total ash content; vit: Vitamin C; Phe: Total Phenolics; Flav: Total flavonoids; Anti: Antioxidant activity.

3.5. Correlation of fruit productivity, fruit quality indices, and health-related compounds

Figure 7 showed a positive correlation among all traits under study. Whereas, fruit drop showed a negative correlation with all other traits. A highly positive correlation was observed among all health-related compounds (Figures 7 and 8).

Figure 7. Correlation coefficient (r) representing the association among fruit productivity parameters, fruit quality indices and health related compounds. Flo: Number of flowers per branch; Set: Fruit set; Dro: Fruit drop; Siz: Fruit size; Wei: Fruit weight; Yie: Yield per tree; Fir: Fruit firmness; Jui: Fruit Juice; Stri: Strife index; TAA: Titratable acidity; TSS: Total soluble solids; Fib: Crude fiber; Ash: Total ash content; vit: Vitamin C; Phe: Total Phenolics; Flav: Total flavonoids; Anti: Antioxidant activity.

Figure 8. Mode of action of GA₃ for controlling pre-mature fruit drop (PFD) in apples.

4. Discussion

The primary goal of this work was to determine the effect of different PGRs on PFD and postharvest quality of three different apple varieties grown under different environmental conditions. Generally, all treatments had a positive effect in minimizing the PFD, however, specifically GA₃ at 25 ppm showed the best results in terms of all parameters studied. In our results, increased number of flowers per branch, high rate of fruit set (%), and reduced fruit drop (%) was noticed in apples which were sprayed with GA₃, which could be due to the modulating role of PGR’s in the mobilization of nutrients in developing organs which can influence the prolonged existence of flowers (Almeida et al., 2004). GA₃ is considered as a growth hormone that usually helps in maintaining the cell wall integrity by increasing anabolism, which results in increased fruit set and reduced fruit drop (Gangadhar et al., 2019). It also helps in increasing the cellulose content of the pedicle which in return strengthens the attachment of fruit and pedicels (Alshallash et al., 2023). Along with all this, GA₃ is also known to reduce the level of endogenous abscisic acid and can effectively reduce PFD (Zhao et al., 2022). Our results showed that ‘Star Crimson’ had higher number of flowers per branch, better fruit set, and less fruit drop at Dheerkot location followed by Bagh location. This might be due to the fact that the growing season temperature influences flower bloom and PFD. Due to the rise in day temperature, the fruits took less time to become mature and remain attached with the pedicle (Raja et al., 2017), while at cooler locations the fruit takes longer time to become mature and due to low temperature the plant could not restore enough energy. Due to this low energy, a reduced photosynthetic rate occurred and resultantly PFD increased (Nievola et al., 2017). In our results, PFD was noticed more at Rawalakot location which could be linked to the extremely low temperature during the growing season as compared to Dheerkot and Bagh locations.

The most promising results were obtained in terms of fruit size, fruit weight, and yield in all apple trees which were sprayed with GA₃. Gibberellins are known for their ability to support cell elongation and cell division, especially at younger stages of growth (Zhang et al., 2023). This increased cell elongation and cell division results in increased fruit weight and fruit size, which ultimately helps in increased yield. It has been shown that spraying of GA₃ on apples can help in increasing fruit weight and reduces the number of asymmetric fruits and improves fruit quality (Liu et al., 2022). In our case, the application of GA₃ gave the best results at all locations irrespective of temperature at different locations, which is a strong evidence that gibberellins effectively regulate abiotic and biotic stress resistance (Zhang et al., 2023). In addition to this, GA₃ is also responsible to reduce the breakdown of cells (Galimba et al., 2019). This transition gene is responsible for 5 folds higher yield expression during fruit growth (Dash et al., 2013). In our results, the low yield reported at Rawalakot location could be linked to low temperature. The results of a previous study indicated that the low values of temperature, especially in winter and spring frost, significantly reduced the apple’s yield (Dalhaus et al., 2020). Furthermore, it could be attributed to the fact that apple trees require a specific temperature to break dormancy as the low temperature is not suitable for releasing dormancy, therefore, forcibly breaking dormancy will greatly reduce their flowering and fruit setting rates and consequently affect the overall yield (Han et al., 2023).

Our results showed that maximum fruit firmness and fruit juice was obtained from the fruits treated with GA₃ at all locations. Fruit juice was positively correlated with fruit size. The results of correlation showed that with 1 unit increase in fruit size, the increase in juice content was 0.84 units. Similar results were obtained in a previous study by Moneruzzaman et al. (2011), who reported that exogenous application of GA₃ increased the fruit size and resultantly juice content were also increased. Low fruit firmness in all apple varieties at Rawalakot location could be due to low air temperature during the months of April and May. This is period when fruit development and cell enlargement occur and at this time the plant needs more water and minerals. Low air temperature reduces the respiration rate which reduces the synthesis of protein, aromatic volatiles, and ethylene, thus results in low growth. Furthermore, nutrient uptake is also complying with respiration rate, which involves lower uptake and partitioning of calcium. Lower growth rate and calcium content of apples both imply lower firmness levels at harvest (Lachapelle et al., 2013).

Fruit quality indices showed that apple trees sprayed with GA₃ had better fruit quality. Whereas, other treatments such as NAA and 2, 4-D resulted in lower fruit quality indices. It was reported that NAA is responsible for the decrease in titratable acidity, starch degradation and high rate of oxidation during respiration and sugar conversion (Abdel-Sattar et al., 2023). In addition, there might be an increase in ethylene biosynthesis when NAA was applied at pre-harvest stage (Li and Yuan, 2008). NAA application not only accelerated the maturation process but also enhanced the ripening process in fruits. Similarly, the process of starch degradation was also accelerated by NAA treatment. Similar results were reported in a previous study who reported that NAA accelerated fruit ripening by reducing the quality of fruits (Trainotti et al., 2007). While GA₃ increased the quality of fruit by delaying the respiration rate and regulating the accumulation of carbohydrates (Yang et al., 2023). In the current study, the fruit quality indices showed lower values at Rawalakot location in case of all parameters studied. This might be due to the fact that low temperature and frost during the flowering season reduced the quantity as well as the quality of apple fruits (Dalhaus et al., 2020). Moreover, Bui et al. (2021) also reported similar findings where they observed that high temperature during the flowering period played a positive effect on the overall quality of apple fruits.

Our results indicated that health-related compounds were also significantly affected by exogenous application of GA₃. It has been observed already that the health-related compounds are highly dependent on variety as well as agro-climatic conditions (Hussein et al., 2023; Zahid et al., 2021). In this regard, a previous report showed that exogenous application of GA₃ increased the vitamin C content by 10.3%–21.2% (Rokaya et al., 2016). Oxidation of vitamin C content is found to influence cell elongation and increase in fruit weight (Smirnoff, 2000). Thus, the increase in vitamin C content might be one of the reasons that GA₃ application increased cell area and therefore, fruit volume and weight (Li et al., 2019). Principal component analysis and correlation matrix showed a positive correlation among vitamin C content, fruit size, and fruit weight. Total phenolics, total flavonoids, and antioxidant activity was also increased with exogenous application of GA₃. These findings are in consistent with the results of Khandaker et al. (2013) who reported that GA₃ significantly promoted the biosynthesis of secondary metabolites in fruits with higher value of antioxidants. It was suggested that exogenous application of GA₃ results in the accumulation of pigments. It also helps in the degradation of chlorophyll which is accompanied by the synthesis of other pigments such as flavonoids during the turning stage (Pourmorad et al., 2006). Our results showed that Dheerkot location was favoured in maintaining the high amount of vitamin C, total phenolics, total flavonoids, and antioxidant activity. Whereas, Rawalakot location showed poor values in terms of health-related compounds as compared to other locations. This might be due to the fact that low temperature at Rawalakot during fruit ripening stage affected the cell turgor and fruit water content which consequently reduced the proportion of secondary metabolites along with antioxidant activity (Table 2; Cervantes et al., 2020). As reported earlier, high temperature and long photoperiods are responsible for vitamin C, phenolics and antioxidant activity in various horticultural crops (Wang & Zheng, 2001). These secondary metabolites such as total phenolics and total flavonoids were positively correlated with antioxidant activity as shown in the principal component analysis and correlation matrix. Health related compounds are indicators for healthy properties of fruits and vegetables and provide help as a tool in the selection of different varieties for breeding programs.

Table 2. Mean maximum and mean minimum temperatures of the experimental sites during year 2022 and year 2023.

Bagh
Months	Year 2021		Year 2022
	Mean maximum temperature	Mean minimum temperature	Mean maximum temperature	Mean minimum temperature
January	27˚C	7˚C	24 °C	12 °C
February	22˚C	4˚C	20 °C	6 °C
March	25˚C	6˚C	24 °C	8 °C
April	33˚C	6˚C	38 °C	18 °C
May	34˚C	17˚C	45 °C	18 °C
June	40˚C	18˚C	46 °C	18 °C
July	34˚C	18˚C	40 °C	24 °C
August	31˚C	15˚C	38 °C	24 °C
September	29˚C	16˚C	37 °C	16 °C
October	28˚C	7˚C	32 °C	8 °C
November	22˚C	7˚C	32 °C	8 °C
December	20˚C	6˚C	30 °C	6 °C
Dheerkot
Months	Mean maximum temperature	Mean minimum temperature	Mean maximum temperature	Mean minimum temperature
January	24˚C	5˚C	23 °C	10 °C
February	19˚C	3˚C	19 °C	7 °C
March	23˚C	5˚C	23 °C	7 °C
April	30˚C	6˚C	36 °C	19 °C
May	31˚C	15˚C	38 °C	17 °C
June	45˚C	17˚C	39 °C	18 °C
July	32˚C	17˚C	38 °C	23 °C
August	29˚C	13˚C	36 °C	24 °C
September	27˚C	14˚C	35 °C	15 °C
October	27˚C	6˚C	30 °C	7 °C
November	21˚C	5˚C	30 °C	6 °C
December	19˚C	4˚C	28 °C	5 °C
Rawalakot
Months	Mean maximum temperature	Mean minimum temperature	Mean maximum temperature	Mean minimum temperature
January	13˚C	−5˚C	11 °C	−4 °C
February	16˚C	−2˚C	14 °C	−4 °C
March	21˚C	1˚C	24 °C	6 °C
April	26˚C	1˚C	30 °C	6 °C
May	31˚C	9˚C	35 °C	8 °C
June	34˚C	14˚C	34 °C	16 °C
July	33˚C	16˚C	35 °C	16 °C
August	27˚C	14˚C	34 °C	16 °C
September	27˚C	13˚C	32 °C	8 °C
October	25˚C	3˚C	30 °C	6 °C
November	19˚C	1˚C	20 °C	−4 °C
December	17˚C	−2˚C	12 °C	−12 °C

5. Conclusions

It can be concluded from this study that exogenous application of GA₃ (25 ppm) before flowering helps in reducing PFD and increases the postharvest nutritional quality of apples. It was also observed that to some extent PFD was dependent on varietal genotypes and environmental variables as well. Moreover, it could be concluded that ‘Star Crimson’ variety of apples showed more health-related compounds with higher amounts of fruit quality indices when exogenously sprayed with GA₃ (25 ppm). In the case of location, Dheerkot and Bagh showed more suitable environmental conditions for growing apples with high yield and less PFD as compared to Rawalakot.

Acknowledgements

Authors would like to acknowledge Higher Education Commission, Pakistan and World Bank for funding this research under Local Challenge Fund (Project No. 20-LCF-229/RGM/R&D/HEC/2020).

Disclosure statement

Mehdi Maqbool current address is Department of Horticulture, Faculty of Agriculture, University of Poonch Rawalakot, Azad Jammu and Kashmir, Pakistan.

Data availability statement

The data have been given in manuscript. If any other details are needed, we will be happy to provide that.

Additional information

Funding

This work was supported by the Higher Education Commission, Pakistan under Local Challenge Fund (Project No. 20-LCF-229/RGM/R&D/HEC/2020).