Assessment of germination dynamics through maturity and pre-treatment studies in Flacourtia indica (burm.f.) Merr.

Introduction

In the pursuit of achieving Sustainable Development Goals (SDGs), particularly the eradication of hunger and malnutrition, marginal fruits have garnered attention for their potential contributions. These fruits, known for their nutritional richness, climate resilience, and resistance to various stresses, also serve as valuable sources of breeding material for crop improvement, making them essential to global food security. One such underutilized species is the Governor's Plum (Flacourtia indica), a tropical and subtropical fruit-bearing shrub or small tree from the Salicaceae family. This resilient plant, native to tropical regions, is prized not only for its tasty fruits but also for its diverse medicinal properties. Its various parts are utilized for therapeutic purposes, reinforcing its reputation as a "nature's pharmacy." Flacourtia indica is highly adaptable, thriving in a variety of environments including coastal areas, lowland dry and humid regions, and riverine habitats (Dunlop et al. 2005) [8] . It is commonly found in tropical dry deciduous and thorn forests, as well as in seasonally dry forests, woodlands, thickets, wooded grasslands, and riparian zones, at elevations ranging from sea level to 2400 meters (Orwa et al. 2009; PIER 2014) [18] . Its hardiness and ability to thrive in suboptimal conditions make it a promising crop for regions with limited agricultural resources. Its drought resistance and adaptability to tropical climates also make it ideal for home gardens and orchards. However, the species faces challenges in propagation, primarily due to poor seed germination rates, often below 20%, and the absence of sustainable replenishment strategies (Prins & Maghembe 1994) [22] . Nondeep physiological dormancy (Gomez et al. 2019) [11] , imposed by the hard endocarp, further complicates propagation efforts. Despite its potential, there is limited scientific literature on the seed biology of Flacourtia indica, particularly regarding seed maturity and germination dynamics. This study seeks to bridge critical knowledge gaps by systematically investigating the criteria and indicators of seed maturity and germination parameters of Flacourtia indica. Employing both qualitative and quantitative methodologies, this research aims to provide a comprehensive understanding of the species' germination dynamics.

Materials and Methods

Study Area and Sample Collection

The study was carried out at Jabalpur, Madhya Pradesh, India, located between 21°17' to 26°52' N latitude and 74°08' to 82°49' E longitude. The region experiences a subtropical climate with hot summers (April-June), monsoon rains (July-September), and cool winters. Annual rainfall averages around 1,370 mm, with temperatures ranging from 10°C in winter to 48°C in summer. Flacourtia indica, naturally distributed in the region, was sampled from mature trees at various developmental stages after anthesis to evaluate seed maturity and germination.

Evaluation of Seed Maturity

Seed maturity was assessed using physical and physiological indicators. Observations were made from the start of flowering to fruit development, with seeds collected between 20 and 100 days after anthesis (DAA). The collected fruits were transported in cotton bags, and seeds were extracted through depulping. Evaluations included: 2.2.1 Dimensions: Fruits and seeds were measured for length and width using a digital caliper. Measurements were taken from three replicates of ten fruits and fifty seeds each.

Weight:

Fresh and dry weights were recorded using an analytical balance. Fresh mass was determined from three replicates of ten fruits and fifty seeds. Dry mass was measured after drying at 80°C until constant weight was achieved. Data were scaled to represent 100 fruits and 1000 seeds.

Color:

Fruit and seed color were recorded using the RHS color chart for 50 samples at each harvest stage.

Germination:

Germination was tested by placing three replicates of 50 seeds each on filter paper in petri dishes at 30 ± 2°C with a 16/8 light/dark cycle (ISTA rules). Germination was assessed daily for 28 days, with seeds considered germinated if radicles reached at least 1 cm. The percentage of viable seeds was calculated, and desiccation sensitivity was also tested. 2.2.5 Vigour Index: Seed vigour was calculated as the product of the mean germination percentage and mean seedling length (Abdul-Baki & Anderson, 1973) [1] .

Evaluation of Seed Germination behavior

Germination trials were conducted under controlled conditions to identify optimal pre-treatment methods and assess the role of the seed coat in inhibiting water uptake. The study utilized a completely randomized design with three replicates of 100 seeds each.

Imbibition assays:

Seeds were weighed on an analytical balance and immersed in 50 mL of tap water in a beaker for 48 hours. At intervals of 1, 2, 4, 6, 8, 10, 12, 18, 24, and 30 hours, seeds were removed, blotted dry, reweighed, and returned to the water. This process continued until the seeds reached a constant weight. Water absorption was estimated from the weight gain, with percentage increase in seed mass (Bewley & Black, 1982) [5] calculated using: Seed mass increase (%) = [(Wi-Wd)/Wd] × 100, Where, Wi and Wd are the mass of imbibed and dry seeds, respectively.

Pre-treatment:

Following pre-treatment methods were employed to find out the suitable pre-sowing treatments for enhancing seed germination (Table 1). Data on seed germination were recorded starting from seed germination till culmination and computed germination-related parameters. Germinationrelated parameters were determined as follows:

• First Day of Germination (FDG) (Kader, 1998) FDG= Dg -Ds • Where, Dg = First germination day and Ds = Seed sowing day. • Last Day of Germination (LDG) (Kader, 1998) (LDG)= Df -Ds • Where, Df = Final germination day and Ds = Seed sowing day. • Time Spread of Germination (TSG) (Kader, 1998) TSG= LDG -FDG • Where, LDG= Last Day of Germination, FDG= First Day of Germination • Final Germination Percentage (FGP) (Scott et al., 1984) FGP= G / T × 100 • Seedling vigour index I= G% X Seedling length (cm) (Abdul-Baki, and Anderson 1973) [1] . • Mean daily germination (MDG) (Czabator, F. J. 1962). MDG = GP / Gd • Where GP = Germination percentage and Gd = No. of days to complete germination.

Statistical Analysis

Data were analysed using ANOVA to test for significant differences in seed size, weight, moisture content, viability, and germination parameters between different developmental stages and pre-treatment methods. Post-hoc tests were conducted where applicable to identify specific differences between groups.

Results

Impact of maturity stages on quality parameters:

The maturity assessment of Flacourtia indica results are discussed under the following head:

Changes in Fruit and Seed Size:

The results of study presented in the Figure 1 showing the growth pattern of fruit and seed dimensions over time, measured in days after anthesis (DAA). The variables observed in the study were fruit length, fruit width, seed length, and seed width. The study observed the growth patterns of fruit and seeds over time. Fruit length (blue line) increased gradually, peaking at 2.13 cm at 90 days after anthesis (DAA), with a notable growth spurt between 70 and 90 DAA before slowing down, likely due to moisture loss and tissue disintegration from the fruit's pulpy nature. Similarly, fruit width (gray line) exhibited significant growth starting around 70 DAA, reaching its maximum at 90 DAA and slightly declining by 100 DAA, suggesting maturity. In contrast, seed length (solid red line) showed a steady increase throughout the period, with a plateau until 70 DAA, while seed width (yellow line) experienced minimal changes after attaining maximum at 40 DAA, reflecting a slower growth rate compared to fruit dimensions.

The graph highlights that both fruit and seed dimensions increase over time, with the most rapid growth in fruit size (length and width) occurring between 70 and 90 days after anthesis (DAA), driven by cell expansion and nutrient accumulation. After 90 DAA, a slight decrease in fruit width suggests the onset of maturation or ripening. Seed growth is steady but slower, without experiencing the same rapid growth phase as the fruit. Pairwise comparisons show significant differences between different stages for both seeds and fruits, emphasizing the contrast in growth dynamics.

Changes in Fresh and Dry Weight of Fruit and Seed

The data presented in the Figure 2 indicate changes in the fresh and dry weights of fruits (mean weight of 100 fruits) and seeds (mean weight of 1000 seeds) over time, measured in days after anthesis (DAA). The fresh weight of fruits (blue line) increased steadily from 20 days after anthesis (DAA), peaking at 83.85 g on 80 DAA before declining, likely due to water loss associated with maturation. In contrast, the fresh weight of seeds (gray line) followed a gradual increase that also peaked around 80 DAA, indicating growth and moisture accumulation. After 80 DAA, seed weight declined slightly, reflecting the onset of maturation.

The dry weight of fruits (red line) steadily increased throughout the observation period, peaking around 80-90 DAA, indicating the accumulation of dry matter. Similarly, the dry weight of seeds (yellow line) showed a gradual increase over time, highlighting the ongoing accumulation of nutrients essential for seed development.

Changes in Fruit and Seed Color

The changes in fruit color are good indicator of fruit maturity.

The fruit remains green from 20 to 60 DAA, then transitions to deep purplish-red at 70 DAA, darkening to red by 100 DAA, indicating ripening. Seeds are not visible at 20 DAA but begin as strong orange-yellow color at 30 DAA, shifting to light yellowish-pink by 60 DAA and remaining stable through 100 DAA, signalling gradual seed maturation (Figure 3).

Seed Germination and vigour:

The study analysed seed germination percentages at different developmental stages (Table 3). Fresh seeds showed no germination from 20 to 60 DAA, with only a low percentage (1.83±3.

Imbibition Assays:

Figure 4 illustrates the changes in seed fresh weight (% increse) during various periods of imbibition time (in hours) for Flacourtia indica seeds, showing a consistent increase over a 30-hour period that indicates effective water uptake. The imbibition process follows a triphasic pattern. In the initial phase (0-8 hours), seed fresh weight increases steadily to approximately 50%, reflecting rapid water absorption as the seeds swell. During the steady rise phase (8-12 hours), fresh weight reaches about 55%, indicating continued active water uptake. After 12 hours, the rate of increase slows, leading to a plateau phase (12-30 hours) where fresh weight stabilizes at around 60% increse by 24 hours, suggesting that the seeds have reached their maximum water absorption capacity. This pattern indicates that imbibition is a rapid process initially, reaching saturation by 24 hours. The fresh weight percentage increase serves as a key indicator of water uptake, which is essential for initiating germination and supporting further developmental processes.

Impact of pre-treatment methods on seed quality parameters:

Table 4 presents the effects of various pretreatments on germination parameters of Flacourtia indica seeds. The first day of germination (FDG) was observed to start on day 4 for treatments T3, T5, T6, T7, T8, T9, and T12, significantly earlier than the control treatments (T0 and T1), which germinated on day 7. The last day of germination (LDG) was for T0 and T1 (18 days) and earliest for T4, T5, T9, T10, and T12 (10 days). Time spread of germination showed that T4, T10, and T12 had the shortest range (3.0-6.0 days), while T0 and T1 had the longest (11 days). The highest final germination percentage (FGP) was found in T9 (47.2%), while T4, T10, T11, and T12 exhibited lower percentages (11-13%)

Discussion

The maturity assessment of Flacourtia indica is critical for determining the optimal harvesting stage of its fruits, which directly impacts seed germination and seedling quality. This knowledge can significantly save time and costs for tree nursery growers by identifying the ideal maturity stage to collect fruits before they fall and deteriorate. As seen in the data, the transition from green to red and eventually dark red in fruit color serves as a reliable visual indicator of maturation. The findings show that both fruit and seed size and weight increase significantly over time, peaking around 80-90 days after anthesis (DAA). This period corresponds with the highest fresh and dry weights, alongside a notable decrease in moisture content, suggesting that seeds collected during this window are most viable for propagation.

In the growth phase (20-80 DAA), substantial increases in fresh weight of both fruits and seeds indicate active growth and water uptake, accompanied by increases in dry weight, reflecting the accumulation of essential dry matter. The maturation phase (80-100 DAA) marks a shift, as fresh weight declines due to moisture loss while dry weight stabilizes, indicating hardening and maturation of the seeds and fruits. [18] . Pre-treatment methods significantly influenced germination rates, with soaking in GA3 at 500 PPM for 24 hours (T9) demonstrating remarkable efficacy. This treatment yielded the highest final germination percentage, seedling vigour, and overall germination performance, corroborating findings from previous studies on the Flacourtia genus (Janick and Paull 2008; Orwa et al. 2009; Gomez et al. 2019) [12] . Treatments involving KNO 3 (T7) and thiourea (T6) also showed improved metrics compared to controls, while mechanical scarification (T5) and soaking in water (T1, T2) yielded positive but lesser effects. Conversely, treatments involving H 2 SO 4 were generally less effective, indicating their unsuitability for Flacourtia indica.

Conclusion

The study highlights the critical importance of maturation timing and pre-treatment methods in enhancing the germination and vigour of Flacourtia indica seeds. Seeds collected at 80-90 DAA demonstrated optimal maturity, physiological quality, and desiccation tolerance. Pre-treatments involving in GA 3 at 500 PPM for 24 hours significantly improved germination performance, making them recommended practices for Flacourtia indica seed propagation and conservation efforts. This information is vital for conservation efforts and can significantly improve propagation practices in nursery settings, thereby supporting sustainable management of this valuable species.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgment

The present work is the outcome of an ongoing project focused on the conservation and sustainable management of wild edible fruiting species, generously sponsored by the CAMPA, Ministry of Environment, Forests & Climate Change, Government of India, New Delhi. We extend our sincere gratitude to this funding agency for their invaluable support.

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