Study Site

The field experiment was conducted at tehsil Shujabad (29.8717° N, 71.3231° E) located in the district Multan province of Punjab, Pakistan. Shujabad is a renowned agricultural land due to its hot weather, fertile soil and irrigation system, such climate makes it favorable for the production of economic crops such as cotton, wheat, dates and mangoes. This experiment was piloted by WWF-Pakistan under the programme Food and Markets, Better Cotton project.

Seed procurement and treatment design:

The cotton seeds of two different high-performing cotton cultivars named IUB^-13 (V1) and FH-Lalazar (V2) were used in the current research work and were procured from Ayub Agricultural Research Institute (AARI), Faisalabad-Pakistan for the purpose to evaluate the effect of different seed priming methods on cotton such as rate of germination, growth and production.

Seed priming treatments:

The seeds of both cotton varieties were treated for seed priming as per the experimental treatment design.

1. For treatment T2 (hydro-priming) seeds were imbibed with water for a limited period, and cottonseeds were soaked for 24 h at room temperature (24 h), drained and then used for sowing.

2. For treatments T3 and T4 (hormonal priming), two different concentrations of indole acetic acid were used (10 mg L^-1), and (20 mg L^-1). Solutions for hormonal priming were prepared by dissolving 10mg and 20mg indole acetic acid in 1 liter of distilled water. Cottonseeds of both varieties were soaked in the solutions for a period of 24 h, seeds were drained and then used for sowing.

3. For treatments T5 and T6 (halo-priming), two different concentrations of potassium nitrate (KNO₃) 3 g L^-1 and 6 g L^-1 were prepared by dissolving 3 g and 6 g of KNO₃ in 1 liter of distilled water, separately. Cotton seeds of both varieties were primed for about 24 h, seeds were drained and used for sowing.

Experimental layout

The experimental area for the field trial was 0.04 hectares (4046.86 m2). The experiment was laid out under RCBD (Randomized complete block design) with three replications for each treatment and the plot size for each treatment was 25ft×25ft. To keep the homogeneity, an almost 5 feet buffer area was kept around the actual experimental area. Cottonseeds (primed and unprimed) were sown in each plot by maintaining plant-to-plant (0.2286 m) and row-to-row (0.762 m) distance. Land preparation practices included rotavator (1-time) and chisel plough (2-times); 2-times plowing and 2-times planking for seedbed preparation and ridges were formed for seed sowing. A uniform dose of urea (100 kg acre^-1) and diammonium phosphate [DAP (50 kg acre^-1)] was applied in the experimental plots. Moreover, two pesticides namely deltamethrin and lufenuron were sprayed for pest management (against white fly and pink bollworm). For irrigation purposes about 10-times, the land was uniformly irrigated. After seed sowing, the rate of seed germination (%) and seedling emergence (3-5 days intervals %) were recorded for both varieties V1 and V2. About five plants from each replication were randomly selected and tagged (90 plants from each cotton variety). Almost 180 plants were randomly selected (from V1 and V2) and tagged from all replications of the above-mentioned six treatments. To record the data from each treatment, the tagged cotton plant was observed and assessed for the attributes related to growth, agronomic traits and yield. The growth attributes included plant height (cm), shoot length (cm) and root length (cm), plant fresh weight (g) and plant dry weight (g); agronomic parameters such as the number of branches plant^-1, and the number of fruiting branches plant^-1 and number of affected fruits and bolls plant^-1; and yield parameters such as the number of bolls plant^-1, boll weight plant^-1 (g) and cotton yield (kg ha^-1). Plant height was measured using a measuring tape from the base to the tip of the plant, and similarly, root and shoot lengths were also measured with measuring tape. Plant fresh weight (g) was noted at the time of crop harvest by using an electric weight balance. To take the dry weight, tagged plants were sun-dried after harvest and then weighed. About 180 cotton bolls were randomly collected from the marked plants per plot to measure the number of bolls and boll weight (g) plant^-1. Similarly, the cumulative yield from each treatment plot was used to compute the maximum cotton yield (kg ha^-1).

Data Analysis

The data of both cotton varieties were statistically analyzed by subjecting to the analysis of variance (ANOVA) and means were compared by the LSD (least significant difference test) at a level of p ≤ 0.05 using statistix 8.1 software.

Results

Seed germination and emergence percentage

Cottonseeds were primed from T2 to T6 according to the treatment design to check the seed priming effect on both cotton varieties (V1 and V2). Results about the rate of seed germination (%) of both cotton varieties (variety^-1 and variety-2) showed that treatment T5 (halo-priming) had a high percentage of seed germination by 83% followed by the rest of the priming treatments (~75%) as compared to the non-primed plants in T1 (Fig. 1). When seedling emergence (%) was observed at three different intervals of time, the highest seedling emergence of variety IUB^-13 (V1) was shown in T5 and T6 by 92% and 89%, respectively after five days of sowing (5DAS) as compared to the plants in the control treatment (T1). Likewise, for the variety FH-Lalazar (V2), the highest seedling emergence was also observed in T5 and T6 after five days of sowing by 94% and 86%, respectively as compared to the control (Fig. 2). It was noticed that seed priming treatment improved seedling emergence and seed germination percentage.

Growth attributes

Results related to growth attributes showed that plant height in treatment T5 (KNO₃ 3 g L^-1) significantly improved by 35% in variety^-1 and by 28% in variety-2 followed by T4 (21%) as compared to their respective control plant (T1). The results of shoot length and root length showed significant improvement with KNO₃ 6 g L^-1 (T6) priming treatment in both varieties (Fig. 3). Shoot length of both varieties improved by 19% and root length also significantly increased by 40-45% as compared to the control plant (T1). While the trend of plant fresh and dry weight is similar in both varieties (V1 and V2), plant fresh and dry weight is significantly high by ~78% in treatment T5 (KNO₃ 3 g L^-1) followed by T4 (indole acetic acid 20 mg L^-1) (60%) as compared to their respective control plant in T1 (Fig. 4).

Agronomic attributes

The results of the agronomic attributes of the cotton crop significantly amplified as compared to the non-primed cotton plants (Fig. 5). 

Cotton plants of seed priming treatment (T5) with 3 g/L of KNO₃ showed a significant rise in the number of branches per plant by 37% as well as the number of fruiting branches per plant by 17% in both varieties (V1 and V2) as compared to their respective control plant (T1). There is a significant deviation in the results of pest-affected flowers and bolls per plant by 2% in T5 and T6 of both varieties as compared to the other treatments and control plants.

Yield attributes

Likewise, regarding the growth and agronomic traits of the cotton crop, the results of yield attributes showed almost the same trend of yield improvement. The number of cotton bolls per plant has significantly augmented by 43% in treatment T5 which is almost 70 cotton bolls per plant as compared to the non-primed plant with almost 50 cotton bolls per plant. Similarly, our assessment regarding halo-priming revealed that T5 showed a significant improvement in boll weight by 47% as compared to the respective control plant in both varieties. Lastly, the cotton yield was assessed in all treatments of seed priming and found a positive impact on the improvement of cotton yield, as T5 showed a significant increase by 16% (777 kg ha^-1) in variety^-1 and improved by 18% (754 kg ha^-1) in variety-2 (T5) as compared to their respective control cotton crop (650 kg ha^-1) in T1 (Fig. 6).

Figures & Tables

In the agriculture production system, seed quality is a primary concern responsible for the germination behavior leading to better crop performance in the field. So, the quality of seed is essential which can not only contribute to high seed germination but can also help plants to grow earlier and uniformly to attain higher yields of crops [15]. Seed priming is an eco-friendly and cost-effective pre-germinative technique, that has been adopted to improve the rate of seedling emergence, germination, plant growth, nodulation and productivity for various crops even under environmental stresses [11, 16, 17]. Our results indicated a uniform and improved germination behavior under different seed priming treatments, where primed seeds might facilitate the germination by the activation of pre-germination enzymes, enhancement of metabolite production, synthesis of protein and osmotic adjustment as compared to non-primed seeds as reported by previous studies [18, 19, 20, 21]. Similarly, Sung et al. [22] reported that the positive impact of primed seeds on germination rate is triggered by cell cycle regulation and cell elongation processes. Likewise, Chatterjee et al. [23] studied that seed priming treatment stimulates the seed with the activation of enzymes and initiates the synthesis of growth-promoting chemicals in various agronomic crops leading to uniform seed germination. Our results related to cotton seed germination behavior are aligned with the previous studies, Fahad et al. [8] Ahmadvand et al. [10], Nazir et al. [24] reported that KNO₃ primed seeds induce nitrate reductase enzyme can lead to an increase in germination and seedling growth by breaking seed dormancy.

The present study indicated that the growth and agronomic traits of the cotton crop were also enhanced significantly by seed priming. Previous studies exhibited that different priming treatment methods improve the morphological attributes of the plant and the activities of defense-related antioxidant enzymes which enable the plant to encounter environmental stresses including biotic and abiotic [15, 25]. Our results revealed the positive impact of priming on plant growth may be due to developed nutrient use efficiency allowing a higher relative growth rate. Although other priming treatments were also effective the impact of KNO₃ was marked. Results of the experiment showed that KNO₃ primed seeds exhibited a more promising effect on the morphological and yield traits of the cotton crop. Potassium (K) and nitrogen (N), both are vital macronutrients, essential for regulating plant developmental processes viz., protection of cell membrane, protein synthesis, enzyme activation, photosynthesis and stomata regulation [26]. Previously it was noticed that priming treatment/s induced stress resistance may be a consequence of improved discrimination for K+ over Na+ nutrition which enhances the uptake of mineral nutrients from the soil [14, 27]. Besides, priming stimulates the effective germination rate and high plant density resulting in high economic yield [20]. Likewise, in the current study along with the improved morphological traits of the crop, improvement in the yield traits also promising. Similar increments in the yield of various crops have been noticed, previously with the adoption of different seed priming techniques [27, 28, 29, 30]. All these results indicated that different seed priming methods had a significant impact on improved plant growth and potential yield. Thus, it can be used in cotton as an effective and economic technique under field conditions for better cotton production. The results presented in this paper revealed that seeds of both cotton cultivars primed with KNO₃ (3 g L^-1) proved to be more successful in improving germination behavior, growth and yield attributes in the field as compared to the non-primed seeds. The present study provides the direction toward further molecular investigation about the mechanism of plant response under the positive effect of seed priming.

This study highlights the significant impact of seed priming which is a sustainable approach to improving the rate of seed germination. Our current findings concluded that both cotton cultivars with the seed priming treatment/s showed a considerable positive effect on germination rate, growth and yield attributes. The findings suggested that intensive research services should pursue to create awareness and accelerate the adoption of this viable and cost-effective strategy in the cotton-growing areas. Besides, the regulatory pathways of seed priming that pose a positive effect on cotton seeds need to be explored with the integration of molecular approaches.

Author Contributions

F.S: Conceptualization and design methodology; Supervise the field trial.
S.A.M: Investigation and Data Curation.
Z.A.A: Analysis and interpretation of the data; Writing-original draft or revising the manuscript critically for important intellectual content. 
L.A.K: Supervisor; Review and editing the draft
A.I: Project administration

Conflict of Interest

The authors have declare that there is no conflict of interest.

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