Physiological responses of sugarcane to combined application of plant growth regulators under moisture stress

Introduction

About 1.9 billion tonnes of sugarcane are harvested annually, making it the world's largest crop by tonnage [1] . Yet sugarcane is sensitive to water deficits during the grand growth phase, when stem elongation and sucrose accumulation peak [2] . Moisture stress triggers stomatal closure, reduces cell turgor, disrupts photosynthetic electron transport, and accelerates leaf senescence all of which lower cane tonnage and sugar recovery [3] . Plant growth regulators (PGRs) offer a management option to mitigate stress effects. Gibberellic acid (GA₃) promotes cell elongation and internode expansion, while cytokinins like benzylaminopurine (BA) delay senescence and sustain chlorophyll levels [4,5] . Abscisic acid (ABA) regulates stomatal behavior and promotes osmolyte accumulation under drought [6] . Most field trials have tested these PGRs individually, but their combined physiological effects in sugarcane under controlled moisture stress remain poorly documented [7] . This research aimed to compare the effects of GA₃, BA, their combination, and ABA on key physiological parameters and yield of sugarcane under repeated moisture stress cycles. The hypothesis was that combined GA₃ + BA treatment would outperform single-PGR applications by simultaneously promoting growth and delaying stress-induced senescence [8,9] .

Research area description

The pot experiment was conducted in the controlled-environment glasshouse at the School of Agriculture, Policy and Development, University of Reading, Reading, United Kingdom (51°27′N, 0°57′W). The glasshouse maintained day/night temperatures of 30/22 °C, photoperiod of 14 hours (supplemental lighting at 400 µmol m⁻² s⁻¹), and ambient relative humidity of 60-70%. The growing medium was a 3:1 mix of sandy loam soil and perlite with pH 6.8 and organic carbon of 1.2%. Single-bud setts of sugarcane variety Co 0238 were sourced from the Sugarcane Breeding Institute's international exchange programme [10] .

Climatic and environmental conditions

During the June 2022 to March 2023 experimental period, external weather at Reading showed mean monthly temperatures ranging from 5.8 C (January 2023) to 21.3 °C (July 2022). Total precipitation for the period was 612 mm, distributed fairly evenly across months. Solar radiation peaked at 18.4 MJ m⁻² day⁻± in July and declined to 3.1 MJ m⁻² day⁻± in December. Inside the glasshouse, conditions were decoupled from external weather: air temperature was maintained at 30/22 C (day/night) using automated heating and ventilation, and supplemental highpressure sodium lamps ensured a minimum photosynthetically active radiation of 400 µmol m⁻² s⁻± throughout the photoperiod. Humidity was regulated between 60-70% by intermittent misting. Soil temperature in pots averaged 26.4 °C during the day.

Experimental design

The experiment used a single-factor completely randomized design with five treatments and four replications, totaling 20 pots. Each pot contained a single sugarcane plant grown from a single-bud sett. Pots were arranged randomly on raised benches within the glasshouse and re-randomized every two weeks to minimize positional effects from light gradients. Stress cycles began at 40 days after planting: each cycle involved 14 days of withheld irrigation followed by 7 days of re-watering to field capacity. Four consecutive stress cycles were imposed over the growing period. PGR sprays were applied to the foliage using a hand-held atomizer (50 mL per plant) one day before each stress cycle commenced. Control plants received an equivalent spray of distilled water with 0.1% Tween-20.

Materials and Methods

Materials

The research was conducted from June 2022 to March 2023 at the University of Reading glasshouse, United Kingdom. Sugarcane variety Co 0238 was grown in 20-L plastic pots filled with sandy loam-perlite medium. GA₃ (>90% purity, Sigma-Aldrich), BA (>98%, Sigma-Aldrich), and ABA (±cis-trans isomer, Sigma-Aldrich) were dissolved in small volumes of ethanol and diluted to working concentrations with distilled water containing 0.1% Tween-20 as surfactant [11] . Treatments included: T 1 (GA₃ at 50 mg/L), T 2 (BA at 25 mg/L), T 3 (GA₃ + BA combined), T 4 (ABA at 10 mg/L), and T 5 (untreated control). PGRs were applied as foliar sprays at 45, 75, and 105 days after planting.

Methods

A completely randomized design with five treatments and four replications (20 pots) was used. Moisture stress was imposed starting 40 days after planting by withholding irrigation for 14day cycles, followed by 7-day recovery periods, repeated four times. PGR sprays were applied one day before each stress cycle. Relative water content (RWC), chlorophyll stability index (CSI), membrane stability index (MSI), and proline content were measured at the end of each stress cycle. RWC was determined by the fresh-turgid-dry weight method [12] . CSI was measured following Murty and Majumder [13] . Proline was estimated by the ninhydrin method of Bates et al. [14] . MSI was assessed by the electrolyte leakage technique. At harvest (240 days), cane yield per pot, number of internodes, and internode length were recorded. Data were analyzed by ANOVA with Tukey's HSD at p<0.05 [15] . The combined GA₃ + BA treatment (T 3 ) consistently outperformed all other treatments. RWC was maintained at 74.8% compared to 62.1% in the stressed control, a difference of 12.7 percentage points. CSI and MSI followed similar patterns, with T 3 recording 81.3% and 76.4%, respectively. Proline accumulation was highest under T 3 (4.67 µmol/g FW), though ABA-treated plants also showed elevated proline (4.12 µmol/g FW). Cane yield per pot under T 3 was 542.1 g, which was 33.8% higher than the untreated control.

Results

Comprehensive interpretation

The combined PGR treatment consistently produced the best physiological and growth outcomes. GA₃ drove cell elongation while BA sustained chlorophyll and delayed senescence, creating a synergistic effect that neither compound achieved alone. ABA improved osmotic parameters but didn't support growth recovery to the same degree.

Discussion

The synergistic effect of GA₃ + BA observed here is consistent with findings by Iqbal et al. [4] , who reported that gibberellincytokinin combinations enhanced drought tolerance in wheat through improved source-sink coordination. GA₃ likely promoted internode elongation by stimulating cell wall loosening enzymes, while BA maintained photosynthetic capacity by preventing chloroplast degradation [5] . The elevated proline in T 3 plants indicates active osmotic adjustment, which helps maintain cell turgor under low water potential [14] . ABA's role was more conservative it enhanced stomatal regulation and proline synthesis but didn't stimulate growth. This aligns with ABA's known function as a "stress hormone" that prioritizes survival over productivity [6] . The membrane stability data support the idea that combined PGRs reduced oxidative damage, possibly by upregulating antioxidant enzymes like superoxide dismutase and catalase, though these weren't directly measured in this research [16,17] .

Conclusion

Combined foliar application of GA₃ (50 mg/L) and BA (25 mg/L) significantly improved relative water content, chlorophyll stability, membrane integrity, and cane yield of sugarcane under repeated moisture stress. This treatment outperformed individual PGR applications and the untreated control. The results support adopting combined PGR sprays as a management strategy for sugarcane in drought-prone areas. Future work should validate these findings under field conditions across multiple sugarcane varieties and stress intensities [18,19] .

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