The Mitigating Effect of Gibberellic Acid (GA3) and Vc on Drought Stress in Cannabis Seed

Hemp (Cannabis sativa L.), an annual herb of the Cannabaceae family, is renowned for its multi-purpose utility, making it a critical economic crop worldwide. Its fibers are used in high-strength textiles and biodegradable packaging; its seeds are rich in omega-3/6 fatty acids and protein, serving as a nutrient-dense food source and animal feed additive; and its non-psychoactive cannabinoid (e.g., cannabidiol, CBD) is a key ingredient in pharmaceuticals and cosmetics [1].

Geographically, global hemp cultivation is concentrated in regions with temperate climates and adequate rainfall. China leads as the world’s largest producer, accounting for over 60% of global output, with major planting areas in Yunnan, Heilongjiang, and Shaanxi provinces—Yunnan alone contributes ~30% of China’s total hemp yield, thanks to its mild weather and fertile soil [1]. Other significant producers include Canada (focused on CBD-rich varieties), France (textile-grade hemp), and the United States (recovering cultivation post-legalization). However, drought poses a universal threat to these regions: in Yunnan, annual rainfall variability has led to 15–20% yield losses in rain-fed hemp fields; in Canada’s Prairie Provinces, spring droughts reduce seed germination rates by up to 25% [1, 6].

1.2 Drought Stress: A Critical Limitation to Hemp Seed Germination

Seed germination and early seedling establishment are the most vulnerable stages of the hemp life cycle, as they directly determine crop stand density and subsequent yield [2]. Drought stress—defined as a shortage of soil moisture below the threshold required for normal physiological processes—disrupts two key mechanisms in this stage:

Osmotic imbalance: Drought reduces soil water potential, preventing seeds from absorbing water (imbibition), a prerequisite for breaking dormancy and initiating germination.
Oxidative damage: Drought disrupts the balance between reactive oxygen species (ROS) production and scavenging, leading to ROS accumulation. This damages cell membranes, denatures proteins, and inhibits enzyme activity—ultimately stunting seedling growth [22, 23].

Traditional strategies to mitigate drought (e.g., irrigation, mulching) are often cost-prohibitive or logistically unfeasible in arid regions. Thus, exploring biostimulants like Gibberellic Acid (GA₃) and Vc has emerged as a cost-effective alternative. Gibberellic Acid (GA₃), a natural plant growth regulator, is known to break seed dormancy by promoting amylase synthesis, which hydrolyzes stored starches into usable sugars; it also repairs drought-damaged cell membranes [10]. Vc (ascorbic acid), meanwhile, acts as an antioxidant, scavenging excess ROS and protecting cells from oxidative stress [13]. While studies have validated their efficacy in crops like tomato and alfalfa [11, 12], research on their application in hemp under drought remains scarce.

1.3 Objectives of the Study

This study aims to: (1) Evaluate the inhibitory effects of 20% PEG-6000-simulated drought on the physiological traits of Yunma 1 and Bama Huoma seedlings; (2) Determine the optimal concentrations of Gibberellic Acid (GA₃) and Vc for mitigating drought stress; (3) Compare the effectiveness of Gibberellic Acid (GA₃) and Vc using a subordinate function method. The outcomes will support the development of practical seed treatment protocols for drought-prone hemp-growing regions.

2. Market Analysis: Drought Impact on Global Hemp Production

Drought’s economic toll on the global hemp industry is substantial, with implications for both supply chains and market dynamics. According to the International Hemp Association (IHA), drought-related yield losses cost the industry over $200 million annually. In China, Yunnan’s 2022 spring drought reduced textile-grade hemp fiber output by 18%, leading to a 12% increase in fiber prices in downstream textile markets. In Canada, the 2021 Prairie drought cut CBD hemp seed yields by 22%, forcing processors to import seeds from Europe at a 30% premium [1].

Market demand for drought-tolerant hemp varieties and stress-mitigation technologies is rising. A 2023 survey of 500 global hemp growers found that 78% prioritize drought resistance when selecting cultivars, and 65% are willing to invest in seed treatments (e.g., GA₃ soaking) if they guarantee a ≥10% yield improvement. This creates a $50 million annual market for biostimulants tailored to hemp, with Gibberellic Acid (GA₃) emerging as a leading product due to its proven efficacy in other crops [6, 12].

Notably, regional differences exist in demand: Chinese growers (focused on fiber and seed) prefer low-cost GA₃ formulations, while North American and European growers (CBD-focused) prioritize organic Vc-based treatments. This segmentation highlights the need for cultivar-specific recommendations—such as the 600 mg/L GA₃ dosage for Yunma 1 and Bama Huoma identified in this study—to meet diverse market needs.

3. Materials and Methods

3.1 Experimental Materials

Two industrial hemp cultivars were used:

Yunma 1: A high-fiber variety provided by the Economic Crop Research Institute of Yunnan Academy of Agricultural Sciences, widely cultivated in Southwest China.
Bama Huoma: A local seed-rich cultivar from the Economic Crop Research Institute of Guangxi Academy of Agricultural Sciences, adapted to subtropical climates.

Reagents included Gibberellic Acid (GA₃, analytical grade), Vitamin C (Vc, analytical grade), and PEG-6000 (for simulating drought). Equipment included a constant-temperature light incubator, UV-visible spectrophotometer, and electronic balance.

3.2 Experimental Design

3.2.1 Seed Soaking Treatment

Seed sterilization: Plump, uniform seeds were disinfected with 70% ethanol for 30 seconds, rinsed 3x with distilled water, and air-dried.
Chemical treatments: Seeds were soaked in four concentrations of Gibberellic Acid (GA₃: 200, 400, 600, 800 mg/L) or Vc (200, 400, 600, 800 mg/L) at room temperature for 8 hours. Control groups included:
- CK₁: No drought (distilled water soaking, no PEG-6000).
- CK₂: Drought stress (distilled water soaking, 20% PEG-6000).
Seed drying: Treated seeds were air-dried for 24 hours to restore viability.

3.2.2 Germination Under Drought Stress

Germination setup: 30 seeds per treatment were evenly placed in 9 cm Petri dishes lined with filter paper, moistened with 15 mL of 20% PEG-6000 (except CK₁, which used distilled water).
Incubation conditions: 3 days of dark culture at 25℃, followed by a 12 h light (25℃)/12 h dark (20℃) cycle. Each treatment had 3 replicates.
Moisture maintenance: 20% PEG-6000 was added daily via weighing to maintain constant water potential.

3.3 Physiological Index Determination

On the 13th day of cultivation, whole seedlings were harvested to measure the following indices (3 replicates per treatment):

Soluble protein content: Coomassie Brilliant Blue G-250 method [15].
Soluble sugar content: Thiobarbituric acid method [15].
POD activity: Guaiacol method [15].
SOD activity: Nitroblue tetrazolium (NBT) method [16].

3.4 Data Analysis

Data were processed using Excel 2010. The subordinate function method was used to comprehensively evaluate drought resistance, with the formula:(X_{u}=left(X-X_{min }right) /left(X_{max }-X_{min }right)) Where X = measured value of an index, (X_{max}) = maximum value of the index across all treatments, (X_{min}) = minimum value. The average subordinate function value (higher = stronger drought resistance) was used to rank treatments [12].

4. Results and Analysis

4.1 Effects of Gibberellic Acid (GA₃) and Vc on Soluble Substances Under Drought Stress

4.1.1 Soluble Protein Content

As shown in Figure 1, 20% PEG-6000 (CK₂) significantly reduced soluble protein content in both cultivars compared to CK₁. Gibberellic Acid (GA₃) and Vc soaking reversed this trend:

Yunma 1: Soluble protein content increased first, then decreased with rising GA₃ concentration, peaking at 600 mg/L (1.8x higher than CK₂). For Vc, the peak occurred at 400 mg/L (1.6x higher than CK₂).
Bama Huoma: The highest soluble protein content was observed at 600 mg/L GA₃ (2.1x higher than CK₂) and 600 mg/L Vc (1.9x higher than CK₂).

Soluble proteins maintain cell osmotic potential and protect enzymes, so their accumulation indicates enhanced drought adaptation [18].

4.1.2 Soluble Sugar Content

Soluble sugars act as osmolytes and ROS scavengers. Drought (CK₂) reduced soluble sugar content in both cultivars, while Gibberellic Acid (GA₃) and Vc soaking increased it (Figure 2):

Yunma 1: GA₃ treatment peaked at 600 mg/L (2.3x higher than CK₂); Vc treatment peaked at 400 mg/L (2.0x higher than CK₂).
Bama Huoma: GA₃ treatment peaked at 600 mg/L (2.5x higher than CK₂); Vc treatment peaked at 600 mg/L (2.2x higher than CK₂).

This confirms that Gibberellic Acid (GA₃) and Vc enhance osmotic adjustment by promoting soluble sugar accumulation [19].

4.2 Effects of Gibberellic Acid (GA₃) and Vc on Antioxidant Enzyme Activity Under Drought Stress

4.2.1 POD Activity

POD decomposes toxic peroxides produced under drought. CK₂ significantly reduced POD activity, but Gibberellic Acid (GA₃) and Vc soaking restored it (Figure 3):

Yunma 1: POD activity peaked at 600 mg/L GA₃ (3.2x higher than CK₂) and 400 mg/L Vc (2.8x higher than CK₂).
Bama Huoma: POD activity peaked at 600 mg/L GA₃ (3.5x higher than CK₂) and 600 mg/L Vc (3.0x higher than CK₂).

4.2.2 SOD Activity

SOD is the first line of defense against ROS. CK₂ reduced SOD activity, while Gibberellic Acid (GA₃) and Vc soaking increased it (Figure 4):

Yunma 1: SOD activity peaked at 600 mg/L GA₃ (2.9x higher than CK₂) and 400 mg/L Vc (2.5x higher than CK₂).
Bama Huoma: SOD activity peaked at 600 mg/L GA₃ (3.1x higher than CK₂) and 600 mg/L Vc (2.7x higher than CK₂).

These results demonstrate that Gibberellic Acid (GA₃) and Vc enhance antioxidant capacity, reducing oxidative damage [22].

5. Discussion

Drought stress disrupts hemp seed germination by inhibiting osmotic adjustment and antioxidant defense systems [2, 23]. This study shows that Gibberellic Acid (GA₃) and Vc seed soaking alleviate these effects through two key mechanisms:

First, Gibberellic Acid (GA₃) and Vc promote the accumulation of soluble proteins and soluble sugars. Soluble proteins maintain cell turgor and stabilize enzymes, while soluble sugars reduce osmotic potential and scavenge ROS—together, they enhance the seedling’s ability to retain water and resist dehydration [18, 19]. The peak at 600 mg/L GA₃ suggests this concentration optimally activates metabolic pathways for osmolyte synthesis, consistent with findings in alfalfa [12].

Second, Gibberellic Acid (GA₃) and Vc upregulate POD and SOD activity. By scavenging excess ROS, these enzymes protect cell membranes from oxidative damage, ensuring normal nutrient uptake and energy metabolism [22]. The higher efficacy of Gibberellic Acid (GA₃) may be attributed to its dual role: in addition to boosting antioxidants, it breaks seed dormancy by promoting amylase activity, accelerating germination even under drought [10].

Cultivar-specific differences were observed: Bama Huoma required 600 mg/L Vc for optimal performance, while Yunma 1 performed best at 400 mg/L Vc. This may reflect genetic variations in Vc metabolism—Bama Huoma, adapted to subtropical droughts, may have a higher threshold for Vc-induced antioxidant activation [1].

6. Conclusion and Recommendations

6.1 Conclusion

This study confirms that 20% PEG-6000-simulated drought inhibits the physiological activity of hemp seedlings. Seed soaking with Gibberellic Acid (GA₃) and Vc effectively mitigates this stress:

Optimal concentrations: 600 mg/L GA₃ for both Yunma 1 and Bama Huoma; 400 mg/L Vc for Yunma 1 and 600 mg/L Vc for Bama Huoma.
Efficacy ranking: Gibberellic Acid (GA₃) > Vc in enhancing drought resistance.

6.2 Practical Recommendations

Cultivar-specific application: For Yunma 1, use 600 mg/L GA₃ or 400 mg/L Vc for seed soaking; for Bama Huoma, use 600 mg/L GA₃ or 600 mg/L Vc.
Application timing: Soak seeds for 8 hours at room temperature, followed by 24 hours of air-drying before sowing.
Market adaptation: In China’s Yunnan province, promote 600 mg/L GA₃ as a low-cost option for fiber hemp; in North America, combine 600 mg/L GA₃ with organic Vc for CBD hemp to meet regulatory requirements.

6.3 Future Research

Further studies should explore the effects of Gibberellic Acid (GA₃) and Vc on hemp’s reproductive stage under drought and validate their efficacy in field trials across diverse arid regions. Additionally, investigating the molecular mechanisms of GA₃-induced drought resistance could inform the development of drought-tolerant hemp varieties.

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