Effect of seed priming on seed germination in Lisianthus (Eustoma grandiflorum Shinn.)

Introduction

Lisianthus (Eustoma grandiflorum), a member of the family Gentianaceae (Reid, 2009) [18] , is native to North America and is predominantly found in the southern United States, particularly in moist meadows spanning Nebraska, Colorado and Texas (Ohkawa et al., 1991) [16] . Over the years, Lisianthus has gained global prominence as an ornamental plant and a highly valued cut flower. The plant typically grows to a height of 50-70 cm and produces 20-40 flowers, blooming mainly during the summer season (Cantor et al., 2013) [6] . It is an herbaceous species with bluish-green foliage and may bear either a single upright stem or branched stems. The flowers, which can reach up to two inches in diameter, are available in a wide range of colours. Beyond its ornamental appeal, Lisianthus has notable medicinal properties, with its flowers and extracts traditionally used for digestive ailments, anti-inflammatory and antifungal purposes, and for reducing fever and treating malaria. Lisianthus is also employed in cosmetics, fragrances, weight-management products, airpurifying formulations and various homeopathic preparations (Sreelatha et al., 2006) [23] . The seeds exhibit cold-induced dormancy (Ecker et al., 1994) [9] and possess tolerance to soil acidity, pathogen infection and high-temperature stress. However, seed size is extremely small approximately 19,000 seeds per gram or 545,000 seeds per ounce making sowing and handling particularly difficult (Rezaee et al., 2012) [19] . Additionally, the species is characterized by slow and often poor germination, with some batches showing little to no germination even under otherwise favourable conditions (Ecker et al., 1994) [9] . Seed germination in Lisianthus is strongly influenced by environmental factors such as temperature, light and moisture. The seeds are photoblastic, requiring light for successful germination, and both suboptimal and supra-optimal temperatures can suppress the process. The physiological dormancy displayed by Lisianthus seeds is mainly associated with incomplete embryo development and the presence of inhibitory substances that restrict International Journal of Advanced Biochemistry Research 2025; SP-9(12): 34-39 metabolic activation during the initial imbibition phase. Consequently, achieving uniform and rapid germination poses a major challenge for commercial growers, particularly under greenhouse and field conditions. The species' extended germination period and uneven seedling emergence further complicate nursery management and transplanting schedules. To address these limitations, several pre-sowing treatments including hormone application, temperature conditioning and chemical priming have been investigated. These treatments aim to break dormancy, enhance enzymatic activity and improve the physiological readiness of seeds for rapid and uniform germination. Among them, seed priming is recognized as a reliable, cost-effective technique that promotes controlled hydration and activates metabolic processes prior to radicle emergence. Seed priming has proven effective in improving germination traits such as seed vigour, uniform seedling emergence and early seedling growth across various species, particularly under stress conditions (Bajehbaj, 2010) [2] . Primed seeds generally demonstrate higher germination percentages, improved reserve mobilization and reduced time to sprouting compared to non-primed seeds (Tabatabaei, 2014) [24] . Considering the germination challenges associated with Lisianthus, the present study was undertaken to evaluate the effect of growth hormones and chemical treatments at different concentrations on seed germination and its associated attributes.

Material and Methods

The present study was conducted during 2024-25 in the Department of Floriculture and Landscaping, College of Horticulture, Mudigere, under Keladi Shivappa Nayaka University of Agricultural and Horticultural Sciences, Shivamogga. Seeds of Lisianthus var. Nozomi White were generously provided by Sakata Seeds, Japan. Then seeds were soaked in aqueous solutions of various priming treatments, including chemicals viz., thiourea (0.2 ppm, 0.4 ppm and 0.6 ppm), potassium nitrate (0.1%, 0.2% and 0.3%) and plant growth regulators viz., gibberellic acid (GA₃ at 500 ppm, 600 ppm and 700 ppm) and Ethrel (15 ppm, 25 ppm and 35 ppm). Seeds and solution were maintained in a 1:2 (w/v) ratio at a constant temperature of 25 ± 1°C for 24 hours.

Germination Assay: After priming, seeds were removed from the solutions, surface-dried and transferred to 9 cm sterile Petri dishes lined with Whatman No. 1 filter paper moistened with 5 ml of distilled water. Incubation was conducted in a germinator for 30 days under a 12-hour photoperiod provided by fluorescent light (40 μmol m⁻² s⁻¹) at 25 ± 1°C. Germinated seeds were counted every 48 hours for 20 days (Millaku et al., 2012) [14] . Seeds were considered germinated upon appearance of a radicle > 2 mm (Sharma and Sharma, 2010) [22] . Primed seeds were later sown in trays filled with a sterile cocopeat: vermicompost (3:1) medium and maintained under controlled conditions to ensure uniform seedling growth for further evaluation at true leaf emergence stages.

Germination percentage (%): Germination test was conducted in glass petri plates on top of a germination paper. Total number of seeds germinated were counted and expressed into percentage.

Germination percentage =

Number of seeds germinated Total number of seeds sown ×100

The percentage of seeds germinated within a fixed early period was recorded.

Germination energy = Number of seeds germinated at first count Total number of seed germinated by test end × 100

Germination value: Germination value was calculated as per Djanvanshir and Pourbeik (1976) [8] .

DGS (Daily germination speed) =

Cumulative germination per cent Number of days since sowing GP = Germination per cent at the end of test and 10 as a constant. N = total number of daily counts, starting from the date of first germination.

Days taken for initiation (DTIG) and completion of germination (DTCG):

Number of days taken for initiation and completion of germination was recorded for each treatment. The seeds were considered as germinated only when the sprouted plumule was found along with the cotyledons.

It is an expression of total germination at the end of test period with time taken to complete germination (Bonner, 1983).

Un-germinated seeds at the end of the test were given value of n+1

Where n = Number of days in the test and these values will be included in the calculation of means.

The percentage of germinated seedlings that remain alive, healthy, and actively growing after a specific period of observation under experimental conditions. It is an important indicator of seedling vigour and the effectiveness of the growth medium or treatment.

Survival rate = Number of surviving seeds Number of Germinated seeds ×100

Vigour index I: For this total length of seedlings were multiplied by total germination per cent (Abdul-Baki and Anderson, 1973) [1] .

Vigor index I = Total seedling length x germination per cent Plumule and radicle length (mm): Length of the plumule and radicle was measured up to two decimal places and presented in mm at the end of the germination test.

First, second and third true leaf pair (days): The emergence of the first, second and third pair of true leaves (not cotyledons) from the seedling were recorded.

The fresh weight and dry weight of the seedlings from the germination test were weighed using electronic balance and expressed in milligram.

Statistical Analysis

Data obtained from the experiment were analyzed using ANOVA, and treatment differences were tested for significance at the 1% level using critical difference (CD) values.

Results and Discussion

Seed companies must maintain high seed quality and longevity to ensure superior product performance. Rapid and uniform field emergence is key prerequisites for maximizing crop yield, quality and profitability. Slow or uneven seedling emergence often results in smaller plants, reduced field stand and increased susceptibility to soil-borne diseases. To mitigate these challenges, seed priming using chemicals and growth hormones has become a widely adopted practice to enhance seed performance. Over the past two decades, seed enhancement through priming has significantly improved growers' ability to achieve reliable germination in both field and greenhouse environments, and many ornamental species have been successfully primed.

Erken and Kaleci (2010) [10] as well as Millaku et al. (2012) [14] reported improved germination in yellow gentian seeds primed with GA₃ at 600 ppm and 1000 ppm, respectively. In the present study, similar results were observed in Lisianthus, where seeds primed with gibberellic acid recorded significantly higher germination (Fig 1) and its attributing characters in Lisianthus (Table 1) compared to other treatments and the control (Plate 1 and Plate 2). The highest germination percentage (97%) occurred with GA₃ at 700 ppm. This improvement may be attributed to enhanced hydrolytic activity and amylase induction, which improve carbohydrate mobilization and energy availability for embryo growth. These findings are consistent with earlier reports by Rouhi et al. (2010) [20] and Zare et al. (2011) [28] . In contrast, Ethrel treatments resulted in lower germination, possibly due to toxic effects at the concentrations used.

Seed priming with GA₃ at 700 ppm also resulted in the highest germination energy (92.15%) and germination value (6.46), followed by GA₃ at 600 ppm (87.26% and 5.56, respectively). Seeds treated with GA₃ germinate more rapidly and uniformly, as higher concentrations promote enhanced radicle protrusion and early seedling vigour. These results agree with Mohammadi et al. (2020) [15] , who reported that GA₃ accelerates germination processes, and with Shah et al. (2018) [21] , who noted that improved speed and final germination percentages contribute to higher germination values.

Seeds primed with GA₃ at 700 ppm and 600 ppm took the shortest time for initiation (6.00 and 6.33 days, respectively) and completion of germination (15.33 and 18.02 days, respectively). Faster initiation may be due to seed coat softening induced by GA₃, reducing mechanical resistance to radicle emergence. Thiourea also enhanced membrane permeability and enzyme activation, supporting faster germination. These findings align with Rouhi et al. (2010) [20] , who reported improved germination in Tulipa kaufmanniana following dormancy-breaking treatments. Further, GA₃ stimulates enzymatic activities such as αamylase, enhancing starch breakdown, while thiourea facilitates reserve mobilization (Sharma and Sharma, 2010) [22] . The lowest mean germination time (6.55 days) was recorded in seeds primed with GA₃ at 700 ppm. Enhanced metabolism, increased protein synthesis (globulins and cruciferin) and improved membrane integrity due to GA₃ priming likely contributed to this improvement (Varier et al., 2010) [26] . Similar reductions in germination time were reported by Erken and Kaleci (2010) [10] in yellow gentian and by Millaku et al. (2012) [14] , who found enhanced MGT in Gentiana lutea with GA₃ and potassium nitrate priming.

Ghaleh-Shahi et al. (2017) [11] also reported reduced germination time in cock's comb (Celosia cristata) with potassium nitrate priming.

Seedling survival was significantly affected by priming treatments. The highest survival rate (80.70%) occurred with GA₃ at 700 ppm, followed by GA₃ at 600 ppm (72.75%). GA₃ likely enhanced nutrient mobilization and reserve use, resulting in vigorous, stress-tolerant seedlings. These findings correspond with previous work by Varier et al. (2010) [26] and Millaku et al. (2012) [14] in yellow gentian. Gibberellic acid also proved highly effective in enhancing seedling vigour. The highest vigour index (1399) was recorded with GA₃ at 700 ppm, due to stimulated cell division, elongation and increased radicle and plumule growth (Fig. 2). Similar results were reported by Bhandari et al. (2022) in China aster, Rouhi et al. (2010) [20] , Baskaran and Misra (2007) [3] in gladiolus and Zahedi et al. (2012) [27] in sweet William.

The data on the effect of priming treatments on seedling characters is presented in Table 2. GA₃ at 700 ppm resulted in the earliest emergence of the first true leaf pair (6.19 days), followed closely by GA₃ at 600 ppm (6.72 days), whereas Ethrel at 25 ppm resulted in substantial delay (25.17 days). These findings agree with Kaya et al. (2006) and Zare et al. (2011) [28] in Ferula asafoetida, who reported faster vegetative initiation due to enhanced metabolic activity. GA₃ also promoted faster emergence of the second true leaf pair, with the earliest appearance at [17] . Priming also significantly affected development of the third true leaf pair. Seeds primed with GA₃ at 700 ppm showed the earliest emergence (34.13 days), followed closely by 600 ppm (35.47 days), indicating strong vegetative vigour (Fig 2). Delayed development in Ethrel treatments (50.42 days) suggests inhibitory effects at higher concentrations, consistent with Chouhan (2018) [7] .

Seedling fresh and dry weight increased significantly with GA₃ priming. Enhanced water uptake, cell elongation and improved mobilization of stored reserves likely contributed to higher biomass. Similar observations were reported by Thakur and Dhatt (2024) [25] in sunflower, Khan et al. (2017) [13] in gerbera and zinnia, reinforcing the role of GA3 and hormonal regulation in promoting seedling growth and biomass accumulation.

Conclusion

The study demonstrated that seed germination and early seedling growth in Lisianthus were markedly enhanced by gibberellic acid (GA₃) priming. Among all treatments, seeds primed with GA₃ particularly at higher concentrationsconsistently exhibited superior performance, including the highest germination percentage, vigour index and mean germination rate, as well as greater plumule and radicle elongation. These results underscore the pivotal role of GA₃ in promoting rapid, uniform and vigorous seedling establishment. Overall, GA₃ priming proves to be an effective, economical and practical pre-sowing treatment for improving Lisianthus seed performance, offering significant benefits for both nursery management and commercial cultivation.

Acknowledgement

AcknowledgementThe authors sincerely thank M/s Sakata Seeds India Pvt. Ltd., Bengaluru for providing the Lisianthus seeds that enabled this research.

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