Pretreatment and storage effects on seed germination of Adenia viridiflora Craib.ABSTRACT:
Adenia viridiflora Craib, a native edible climbing plant of northeastern Thailand, has considerable potential as a nutrient-rich indigenous vegetable and a promising agricultural crop. Despite its importance, information on seed propagation and storage responses remains poorly understood. This study assessed five pretreatment methods on germination success: control (untreated), mechanical scarification, hot water, gibberellic acid (GA₃), and sulfuric acid (98% H2SO4) under two initial seed moisture contents (10-11% and 48-50% RH). Seeds were kept at room temperature (RT), 5 °C, and -20 °C for a maximum of 14 months to evaluate their longevity. GA₃ pretreatment resulted in the highest germination (>90% in both humidity regimes) with a short mean germination time (12-13 days), and the most synchronized seedling emergence. Hot water moderately improved germination, while mechanical scarification was least effective. Storage tests confirmed orthodox seed behavior. Seed viability remained above 80% for up to 10 months and above 50% after 14 months, with RT and 5 °C conditions better maintaining germination potential compared with -20 °C. These findings demonstrate that A. viridiflora can be successfully propagated with GA₃ pretreatment and conserved via conventional seed banking. RT storage represents a practical and low-cost option for short- to medium-term conservation, thereby supporting the development of climate-resilient crops and long-term genetic resource preservation.
Index terms:
Adenia; germination index; gibberellic acid; seed bank; seed quality
RESUMO:
Adenia viridiflora Craib, uma trepadeira comestível nativa do nordeste da Tailândia, possui considerável potencial como hortaliça rica em nutrientes e cultura agrícola promissora. Apesar de sua importância, as informações sobre a propagação de sementes e suas respostas ao armazenamento ainda são escassas. Este estudo avaliou cinco métodos de pré-tratamento quanto ao sucesso da germinação: controle (sem tratamento), escarificação mecânica, água quente, ácido giberélico (GA₃) e ácido sulfúrico (98% H₂SO₄) sob dois teores iniciais de umidade das sementes (10-11% e 48-50% UR). As sementes foram mantidas à temperatura ambiente (TA), 5 °C e -20 °C por um período máximo de 14 meses para avaliar sua longevidade. O pré-tratamento com GA₃ resultou na maior germinação (>90% em ambos os regimes de umidade), com curto tempo médio de germinação (12-13 dias) e emergência mais sincronizada das plântulas. A água quente melhorou a germinação moderadamente, enquanto a escarificação mecânica foi a menos eficaz. Os testes de armazenamento confirmaram o comportamento ortodoxo das sementes. A viabilidade das sementes permaneceu acima de 80% por até 10 meses e acima de 50% após 14 meses, com as condições de temperatura ambiente e 5 °C mantendo melhor o potencial de germinação em comparação com -20 °C. Esses resultados demonstram que A. viridiflora pode ser propagada com sucesso com pré-tratamento com GA₃ e conservada por meio de bancos de sementes convencionais. O armazenamento em temperatura ambiente representa uma opção prática e de baixo custo para conservação a curto e médio prazo, apoiando assim o desenvolvimento de culturas resilientes ao clima e a preservação de recursos genéticos a longo prazo.
Termos de indexação:
Adenia; índice de germinação; ácido giberélico; banco de sementes; qualidade de sementes
INTRODUCTION
Many edible herbs traditionally used in Thai cuisine are uncultivated and collected directly from their natural habitats, suggesting deep-rooted ethnobotanical traditions (Punchay et al., 2020). Among these is Adenia viridiflora Craib (Passifloraceae), often known in Thai as “Pak E-noon”. It is a perennial climber plant native to northeastern Thailand, Cambodia, Laos, and Vietnam (POWO, 2025). Its young shoots, leaves, flowers, and immature fruits are traditionally consumed between March and August, adding to local diets, supporting rural livelihoods, and enriching regional food culture (Wannasaksri et al., 2021a). Nutritional analyses further indicate that mature leaves of A. viridiflora contain protein, fiber, vital minerals, antioxidants, and enzyme inhibitors that have been linked to diabetes, Alzheimer’s disease, obesity, and hypertension (Wannasaksri et al., 2021a, b). These properties suggest its potential as a nutrient-dense, regionally adaptable functional food crop in Thailand. Promoting natural plants like A. viridiflora for cultivation necessitates a thorough grasp of their seed biology. According to Kildisheva et al. (2020), breaking dormancy and developing species-specific germination techniques are essential steps in enabling proliferation and long-term conservation of underutilized native species. Research on seed germination and storage behaviors is governed by a complex interplay of physical and biological factors. Importantly, such studies provide indispensable baseline information for establishing effective propagation and conservation protocols (Araujo et al., 2017; Gentil et al., 2018; Jose et al., 2018). Despite its potential, the agricultural application of A. viridiflora has been limited due to a lack of understanding of its propagation biology, including seed dormancy mechanism and germination needs.
Pretreatment techniques such as hot-water soaking, mechanical scarification, and sulfuric acid treatment is commonly used to increase water uptake and break dormancy in hard-coated seeds, including Leucaena leucocephala (Lam.) de Wit) and Jathopha curcas L. (Jose et al., 2018; Koobonye et al., 2018) and Neonotonia wightii (Wight & Arn.) J.A.Lackey (Reina-García et al., 2024). Hydropriming and hormopriming with gibberellic acid (GA₃) have the potential to increase germination and seedling strength in favorable and stress conditions. Adhikari and Subedi (2022) reported that GA₃ priming has a significantly effect to increase germination and primary growth of Zea mays L. under drought stress. The effect of chemical priming agents to promote germination and stress tolerance in a variety of crop plants was supported by recent research (MacDonald and Mohan, 2025). GA₃ is an efficient dormancy-breaking agent in several species, by activating α-amylase, decreasing endogenous abscisic acid (ABA) levels, and improving embryo growth (Finch-Savage and Leubner-Metzger, 2006). It helps break dormancy by controlling hormones and starting up metabolic processes. Previous reports show that GA₃ decreases ABA sensitivity and upregulates GA biosynthetic genes like GA3ox and GA20ox while inhibiting ABA catabolic regulators like CYP707A (Liu and Hou, 2018). Transcriptomic analyses in Suaeda glauca (Bunge) Bunge and Arabidopsis thaliana (L.) Heynh. also indicated upregulation of the starch-mobilizing genes (BAM, HXK2, AGLU), implying increased carbohydrate mobilization during germination (Wang et al., 2024; Zhu et al., 2024).
Chen et al. (2024) conducted metabolomics studies in Brassica napus L. and found that ABA-responsive pathways inhibit germination by downregulating starch and sucrose metabolism. In contrast, GA₃ activated metabolic processes that facilitate germination. For Oryza sativa L., seeds soaking in GA₃ enhanced germination rates (Sukifto et al., 2020) and alternating cold stratification with GA₃ treatment increased seedling vigor in Magnolia biondii Pamp. (Chen et al., 2025). Applications in ecological restoration also demonstrated that slow-release GA₃ coatings significantly enhanced emergence rates in Penstemon species without affecting subsequent growth (Larson et al., 2023). More generally, GA₃ has been recognized as an effective hormone for promoting germination uniformity, particularly in species with physiological dormancy (Afzal, 2023). These studies demonstrate GA₃’s conserved and adaptive function in promoting seedling establishment and overcoming dormancy barriers in various plant species.
Seed storage behavior is essential part of conservation strategy. Orthodox seeds can be dried and stored at low temperatures and are therefore amenable to seed banking, whereas recalcitrant and intermediate seeds require alternative strategies such as cryopreservation or field gene banks (Walters, 2015). The FAO Genebank Standards (2014) and the Ellis-Roberts seed viability equations are practical tools for developing storage techniques and estimating seed longevity (Ellis and Roberts, 1980). However, the importance of genetic, physiological, and environmental aspects in seed aging, were importantly considered for species-specific conservation strategies (Pirredda et al., 2024).
Due to highly potential economic plant of A. viridiflora, seed propagation is needed for agriculture and seed storage is needed for conservation. Thus, this study was conducted by comparing five pretreatments (control, mechanical scarification, hot-water soaking, GA₃ soaking, and concentrated H₂SO₄ treatment) under two moisture regimes and assessing seed longevity at room temperature (RT), 5 °C, and −20 °C for up to 14 months. By connecting effective dormancy-breaking treatments with determined storage behavior, these findings aim to provide practical strategies for seedling propagation, seed distribution, and ex situ conservation of this underused species.
MATERIAL AND METHODS
Seed collection and storage
Mature fruits of A. viridiflora, which turn orange and have 3-4 segments, were collected from the Plant Genetic Conservation Project under the Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn-Suranaree University of Technology (RSPG-SUT). The seeds are small, ovoid to ellipsoid, black, measuring about 0.2-0.5 cm in length (Figure 1). Mature seeds then were rinsed thoroughly with clean water, and non-viable seeds (i.e., those that floated) were discarded.
Botanical characteristics of Adenia viridiflora. Habit (A), male flower (B), young fruit and seeds (C), fruits (D), female flower (E), mature seed (F), and longitudinal sections of seed (G).
Following cleaning, the seeds were shade-dried for a period of three days. Once fully dried, seeds were collected and prepared for storage at ambient room temperature. Two seed lots were prepared under different moisture and storage conditions: 1) Low-humidity storage: Seeds were placed in airtight containers with silica gel desiccant and equipped with a humidity indicator. The relative humidity (RH) within the containers stabilized at 10-11%, temperature (approximately 30 °C), and 2) Ambient-humidity storage: Seeds were placed in mesh bags and stored at room temperature (approximately 30 °C) under ambient conditions, with a measured RH of 48-50%.
Experimental design and pretreatment methods
The seeds from different moisture and storage conditions were exposed to five pretreatment methods, 1) T1-Control, untreated seeds , 2) T2-Mechanical scarification, seed coat nicked with a scalpel opposite the micropyle, 3) T3-Hot water, seeds soaked in water at 60 °C for 24 hours, 4) T4- Gibberellic acid (GA₃) treatment, seeds soaked in 500 ppm GA₃ solution for 30 minutes, and 5) T5- Sulfuric acid, seeds immersed in 98%-H₂SO₄ for 4 minutes, followed by thorough rinsing under running water for 15 minutes. Each treatment contained 25 seeds which were replicated four times. So, each treatment had a total of 100 seeds. Seeds in each treatment were put into Petri dish plates with three layers of moist filter paper and incubated in 16/8 hours light/dark at 25 °C.
Seed bank
Seed viability was evaluated at storage periods of 0, 2, 4, 6, 8, 10, 12, and 14 months under three temperature conditions: room temperature (RT, 30 °C), refrigeration (5 °C), and freezing (-20 °C). Seeds with a relative humidity (RH) of 10-11% were used in this experiment. The germination method applied was selected based on the highest germination percentage obtained from the pretreatment experiment, in which seeds were treated with 500 ppm GA3 for 30 minutes.
Data analysis
Germination percentage (GP) was evaluated for the viability of a population of seeds. The equation to calculate germination percentage formula as follows (Seng and Cheong, 2020):
The speed of germination, indicated as germination index (GI), was calculated using the following formula (Jia et al., 2022):
Mean germination time (MGT) is expressed in days, and a lower MGT value indicates faster and more uniform germination, which reflects the effectiveness and quality of the seed germination enhancement treatment. It was calculated using the following formula (Seng and Cheong, 2020):
Where Gi: number of seed germinated on each day, ti: time from start of the experiment.
Mean germination rate (MGR) is calculated as the reciprocal of MGT and is expressed in per day. A higher MGR value indicates faster germination, making it a useful index for comparing the effectiveness of different seed pretreatments or storage conditions.
T50 mention to the time to emergence at 50% of the total germinated seeds emerge during a germination test, making it a useful metric for evaluating seed vigor and the effectiveness of pre-germination treatments (Amin et al., 2024).
Where N: final number of seeds emerged, nj and ni are the cumulative numbers of seeds emerged after adjacent counts during tj and ti, when ni < N/2 > nj.
Germination percentage (GP), germination index (GI), mean germination time (MGT), and mean germination rate (MGR) were subjected to analysis of variance (ANOVA) using SPSS 23 software (IBM Corp., Armonk, NY, USA). Treatment means were compared at a significant level of p<0.05 using Duncan’s multiple range test (DMRT) to determine significant differences among treatments.
RESULTS AND DISCUSSION
Seed germination in Adenia remains poorly reported. However, within the same family, Passifloraceae, seeds of Passiflora spp. have been reported to exhibit dormancy (Ghosh et al., 2017).
The germination responses of Adenia viridiflora seeds to different pretreatments under two initial seed moisture contents are presented in Figure 2 and Table 1). Germination began between 6 and 10 days after sowing across all treatments, consistent with tropical perennial species whose seeds typically require a short metabolic reactivation phase after imbibition (El-Maarouf-Bouteau, 2022). Among all treatments, GA₃ (500 ppm for 30 minutes; T4) produced the most pronounced improvement. In the low-humidity group (10-11% RH), germination percentage (GP) reached 99.0%, while in ambient-humidity seeds (48-50% RH), GP was 92.0%, although the difference was not statistically significant. Germination index (GI) was also highest (2.17 and 2.05, respectively), mean germination time (MGT) was short (10-13 days at low humidity), and time to 50% germination (T50) was reduced by 3-4 days relative to controls. These improvements of GP and GI were statistically significant compared with all other treatments (p < 0.05). The results align with recent work showing that exogenous GA₃ can markedly reduce MGT and increase uniformity in species with physiological dormancy (Luo et al., 2025). Field studies on restoration for several Penstemon species have also reported very large increases in emergence when seeds were coated with GA₃ and sown in favorable microsites (Johnson et al., 2023). GA₃ enhances α-amylase synthesis in the aleurone layer, mobilizing starch reserves for radicle growth and antagonizing ABA-mediated dormancy (Finch-Savage and Leubner-Metzger, 2006; Wang et al., 2024).
Germination performance of Adenia viridiflora seeds under different pretreatments. Cumulative germination trend 10-11% RH (A), Cumulative germination trend 48-50% RH (B), GP (C), GI (D), MGT (E) and T50 (F). Error bars are ±SE.
Hot-water treatment (T3) produced moderate improvement, with GP of 72-75% and GI of 1.58-1.61. The likely mechanism is partial disruption of the seed coat, increasing water uptake and leaching inhibitors, as observed for several semi-domesticated leafy greens and grain Amaranthus species where GA₃ or thermal/chemical scarification improved emergence (Tapfumaneyi et al., 2023). However, prolonged or excessive heating risks thermal damage, explaining why germination did not reach GA₃ levels. Sulfuric-acid scarification (T5) gave intermediate results (37-66% GP), reflecting species-specific responses. Acid scarification effectively removes impermeable seed coats, but excessive exposure can damage embryos and reduce viability (Rego et al., 2011). In contrast, a study on seed germination of Passiflora edulis Sims reported that pretreatment with sulfuric acid for 4 minutes resulted in the highest germination percentage (Ghosh et al., 2017). Mechanical scarification (T2) was least effective, with only 17-26% GP and delayed emergence (0.57-0.7 GI), consistent with reports that mechanical damage can injure embryos when not carefully applied in P. edulis (Alva-Obregon et al., 2014). Control seeds (T1) germinated at 62-73%, indicating that some seeds are non-dormant at harvest.
The effects of storage time and temperature were analyzed, and the results showed that the seed storage time significantly affected GP, GI, MGT and MGR (p <0.05). Storage temperature alone did not have a statistically significant effect, similarly its interaction with time had no statistically significant impact (Table 2). These findings suggest that the physiological integrity of seeds is more sensitive to prolonged storage than to the specific conditions within the tested range.
Storage performance over 14 months is shown in Figure 3 and Table 3. Seed storage duration and temperature showed significant effects on the viability and vigor of A. viridifora (Figure 3). At 0 months, freshly harvested seeds displayed 99.00% germination and high germination index (GI, 2.17) and relative short mean germination time (MGT) and T50, indicating strong initial viability and rapid germination capacity. However, viability declined progressively with prolong storage over 14 months across all storage conditions, suggesting an aging-related deterioration. By 2-6 months, GP dropped significantly across all storage conditions, with the most pronounced reductions observed in seeds stored at -20 °C (Figure 3A). In contrast, seeds at room RT and 5 °C retained relatively higher GP during this period, though overall vigor, as indicated by GI, was reduced (Figure 3B). This suggests that while seeds remain capable of germination, the metabolic efficiency and speed of germination were compromised under extended storage. Notably, both GP and GI exhibited a transient recovery at 8-10 months. However, this improvement was not sustained, as both GP and GI declined markedly by 12-14 months, especially under -20 °C conditions, suggesting partial sensitivity of A. viridiflora seeds to ultra-low temperatures. Such responses have been reported in other tropical taxa, where freezing damages cellular membranes or induced ice crystal formation despite low seed moisture content (Berjak and Pammenter, 2013). At 14 months, seeds stored at RT showed a significant decline in GP to 69.0%, similarly to seeds stored at 5 °C (71.0%) and −20 °C (64.0%) and no significant difference was detected among all conditions (p > 0.05).
Germination performance of Adenia viridiflora seeds under different times and temperature. GP (A), GI (B), MGT (C) and T50 (D). Error bars are ±SE.
MGT and T50 generally increased between 2-6 months across all treatments, reflecting slower germination, but declined at 8-10 months. By 12-14 months, variability increased but did not differ significantly among storage temperatures (Figures 3C and 3D). The ability of A. viridiflora seeds to tolerate drying to 10-11% RH and maintain high viability at low temperatures indicates orthodox seed storage behavior (Walters, 2015). These patterns are consistent with broad seed-storage theory; lowering seed moisture content and temperature exponentially slows ageing (Ellis and Roberts, 1980), and medium-term and long-term storage studies on various forage and legume genera show markedly better maintenance of viability under cool storage than under ambient conditions (Ellis et al.; 2018; Nagel and Börner, 2010). Seeds of A. viridiflora showed slightly better stability at 5 °C than at −20 °C. This aligns with studies reporting that seed survival and quality can be better maintained at 5 °C than at −20 °C, depending on species, moisture content, and storage duration. For example, seeds of Hosta sieboldiana (Hook.) Engl. with 5-10% moisture lost viability more rapidly at −20 °C than at 5 °C (Kanazawa et al., 2015), and black walnut seeds stored at 5 °C with low moisture content (5%) retained germination capacity better than those stored at −20 °C, particularly when vacuum-packaged to reduce oxygen exposure (Flores et al., 2017). Orthodox behavior enables cost-effective gene bank conservation at cold temperatures (5 °C or −20 °C). Based on the results obtained, seed batches stored for up to 12 months still have the potential to produce vigorous seedlings capable of adapting to field conditions. In conservation settings, routine viability monitoring remains advisable at intervals of approximately 3-5 years (FAO, 2014). Overall, the combined evaluation of GP, GI, MGT, and T50 demonstrates that while A. viridiflora seeds are initially viable, they exhibit rapid deterioration under prolong storage. These findings highlight the importance of optimizing seed banking strategies for this endemic species, with storage at room temperature emerging as the most practical and cost-effective option for short to medium term conservation.
CONCLUSIONS
This study provides the first comprehensive evidence that Adenia viridiflora seeds respond strongly to GA₃ pretreatment and exhibit orthodox storage behavior, enabling efficient propagation and long-term conservation. Gemination potential was better maintained at RT and 5 °C conditions than at -20 °C, with RT offering the most practical and cost-effective option for short to medium term storage. These findings establish a foundation for seed biology for this underutilized native species and offer practical protocols for its integration into sustainable agriculture.
ACKNOWLEDGMENTS
This work was supported by Suranaree University of Technology (SUT), Thailand Science Research and Innovation (TSRI) and National Science, Research and Innovation Fund (NSRF) (NRIIS number 195656). The authors thank Chonlathon Pokaew for field support and Thotsaporn Channokkhun for assistance with photography. We are also grateful for the Plant Genetic Conservation Project Under the Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn-Suranaree University of Technology (RSPG- SUT) for their facilitation and support.
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Additional data will be made available by the authors upon reasonable request.
