Seed germination and storage conditions of Ilex embelioides Hook.f. (Magnoliopsida: Aquifoliales: Aquifoliaceae) , a threatened northeastern Indian species

: Ilex embelioides Hook.f. is a threatened tree species endemic to northeastern India. The species propagates naturally through seeds but shows a brief dormancy period. The present study was carried out to assess the germination behaviour, and identify the suitable storage conditions of I. embelioides seeds. Results revealed that warm stratification for 30 days could effectively break dormancy. Seeds pre-treated with gibberellic acid (GA 3, 2,000 mg L -1 ) showed highest germination (63.89 ± 0.91%) as compared to other concentrations of GA 3 as well as potassium nitrate (KNO 3 ). Highest in vitro seed germination percentage (65.56 ± 2.92%) was recorded on (Murashige and Skoog) MS medium containing 10 mg L -1 GA 3 . The viability of the seeds declined with storage period irrespective of its storage condition. After 30 days, highest viability (54.72 %) was observed in seeds stored in moist sand at 25°C. Therefore, application of GA 3 and warm stratification (25 ± 1°C) along with a substrate (moist sand) for 30 days may be considered as ideal conditions for effective germination and storage of I. embelioides seeds. This study can also be used for mass propagation of the species for reintroduction in the wild.

ISSN 0974-7907 (Online); ISSN 0974-7893 (Print)  (Haridasan & Rao 1985;Lasushe et al. 2022) and 'Vulnerable' species of northeastern India (Mir & Upadhaya 2019). It belongs to Aquifoliaceae (holly) family which comprises of more than 500 species distributed throughout temperate and tropical regions of the world (Galle 1997;Cuenoud et al. 2000). In India, the genus Ilex consists of 26 species (Das & Mukherjee 2017). Ilex embelioides is a small tree attaining a height of 10 m and is restricted to primary subtropical and tropical forests. The flowering period in the species was observed during rainy season (April-October) and the fruiting occurs during winter season (mid November-January). The seeds remain dormant for about three months (January-March). Seed germination occurs during March-April with the onset of rain (Upadhaya et al. 2017).
The genus Ilex has small seeds, globular to ovaloblong rudimentary (underdeveloped but differentiated) embryos that grow inside the seed before the emergence of radicle (Martin 1946;Hu 1975;Baskin & Baskin 2004). The embryo in Ilex seeds has low growth potential (physiological dormancy), and warm and/or cold stratification treatments are required to overcome dormancy (Tsang & Corlett 2005;Tezuka et al. 2013). A number of studies have been carried out to understand seed germination and dormancy in other species of Ilex such as I. paraguariensis (Sansberro et al. 2001;Souza et al. 2022), I. uaranine, I. brasiliensis, I. brevicuspis, I. uaran var. uaranine, I. paraguariensis var. paraguariensis, andI. theezans (Galindez et al. 2018). However, there is no information on seed germination of I. embelioides, which is now facing the threat of extinction due to habitat destruction, overexploitation for use as poles and fuel wood, poor regeneration and possibly climaterelated physiological stress (Singh et al. 2023). In order to prevent the species from extinction, mass propagation of the species through seed germination is required (Iralu et al. 2019). Therefore, the present study was carried out to assess the germination behaviour, and identify the suitable storage conditions of I. embeloides seeds. Also in vitro seed germination was conducted to identify optimal conditions for germination.

MATERIALS AND METHODS
Matured fruits of I. embelioides (Image 1A) were collected from two matured individuals (>5 cm diameter at breast height) from Umtong (25.4124 0 N & 92.0034 0 E) and five individuals from Laitryngew (25.2255 0 N & 91.5475 0 E) during the month of December 2021. Seeds were separated from the berries and thoroughly washed with water for 10-20 min to remove the pulp. Healthy seeds were separated from damaged or dead seeds using floatation methods (Pipinis et al. 2011). The seeds were disinfected with 75% ethanol for 30 s, and immediately rinsed with sterile water and dried with absorbent paper. The cleaned seeds were stored at room temperature (24 ± 2 o C) and later used for various experiments.

Moisture content
To estimate the moisture content, three replicates of 50 seeds each were oven dried at 80°C for 24 h and the final mass was noted. Moisture content was determined following ISTA (2008) as follows: M c (%) = (W 2 -W 3 /W 2 -W 1 ) x100 Where, M c is the moisture content, W 1 is the weight of the container, W 2 is the weight of the container with seeds before oven drying and W 3 is the weight of the container with seeds after oven drying.

Effect of gibberellic acid (GA 3 ) and potassium nitrate (KNO 3 ) on seed germination
Plant growth regulator viz., GA 3 , and KNO 3 were used in different concentrations to enhance the germination percentage and reduce the germination time following the protocol as adopted by Iralu & Upadhaya (2016, 2018 and Borah et al. (2023). The different treatments include: (i) control (without GA 3 or KNO 3 ), (ii) seeds soaked in different concentrations of GA 3 i.e., 200, 500, 1,000, 2,000, 4,000 mg L -1 for 48 h, (iii) seeds soaked in different concentrations of KNO 3 i.e., 0.5%, 1%, 1.5% and 2% for 48 h and (iv) seeds soaked in different concentration of GA 3 for initial 48 h and transferred to KNO 3 and kept for another 48 h.
The treated seeds were placed in plastic petri dishes over moist filter paper and kept for germination at 25 ± 1°C. Germination was monitored regularly for a period of 60 days and a seed was considered as germinated with the emergence of a radicle of 1 mm size (Vera et al. 2010). For each replicate in each treatment, the germination percentage was calculated as G = (n/N) × 100, where n is the number of germinated seeds and N is the total number of seeds. Also, the mean germination days were calculated using the formula: Where, n1, n2, n3…. are the number of days taken by individual seeds to germinate and N is the total number J TT of seeds (Ellis & Roberts 1981).

In vitro seed germination
For in vitro seed germination, the seeds were scarified and soaked in distilled water for 24 h, then washed with 2% sodium hypochlorite for 5 min under aseptic condition, and placed on MS medium (Murashige & Skoog 1962) containing 3% sucrose, vitamins and different concentrations of GA 3 (1 mg L -1 , 5 mg L -1 , 10 mg L -1 and 20 mg L -1 ). The cultures were maintained at 25 ± 2°C under a 16/8 h photoperiod (flux of 50 μmol m -2 s -1 ) of cool white fluorescent lights and in the dark. Germination was monitored for a period of 60 days and the germination percentage was calculated.

Effect of stratification on seed germination
To evaluate the effect of warm and cold stratification on seed germination, three replicates of 30 seeds each were packed in sealed polythene bag containing moist sand and stored at 5 ± 1°C in darkness and in an incubator at a constant temperature of 25 ± 1°C for 0 (control), 10, 20, 30, 40, and 50 days. After each treatment, the seeds were thoroughly washed under tap water and incubated in light at 25 ± 2°C and germination was monitored regularly for a period of 60 days.

Seed viability and storage
To understand the seed viability characteristics, three tests were conducted, viz., seeds (approximately 200 seeds/bag) were stored in airtight containers at room temperature (24 ± 2°C and 65% humidity); seeds were packed in airtight polybags and stored at 5 ± 1°C. In the third and fourth test, moist sand was added and the seeds were stored at a constant temperature of 25 ± 1°C and 5 ± 1°C, respectively. The seeds stored in each of the above conditions were retrieved after 10, 20, 30, and 40 days and the viability test was carried out using the Tetrazolium assay (Enescu 1991).

Data analysis
To compare the effect of different concentration of GA 3 and KNO 3 on seed germination, and different storage conditions on viability of seeds, analysis of variance (ANOVA) followed by Tukey's least significant difference test (p <0.05) was done using Origin Pro 2016.

RESULTS AND DISCUSSION
The moisture content of fresh seeds when dried for 24 h in oven at 80°C decreased by 46.99 ± 0.64%.

J TT
A decline in moisture content over a short period of time revealed intermediate nature of seeds (Hong & Ellis 1996;De Vitis et al. 2020). Seed moisture of 10-40% is often considered as desirable for retaining seed longevity in many species (Hampton & Hill 2002;Dadlani et al. 2023).
Plant growth regulator played a major role in breaking seed dormancy in I. embelioides. Seeds treated with 2,000 mg L -1 GA 3 showed highest germination percentage (63.89 ± 0.91%) and the mean number of days required for germination (16.22 ± 0.87) was also reduced (Image 1B). The use of GA 3 increased the germination percentage of I. embelioides during the first four-eight weeks. This result was similar to that Though KNO 3 is widely used chemical for enhancing seed germination (Agrawal & Dadlani 1995), it was not very effective in breaking seed dormancy of I. embelioides (Table 1). However, when KNO 3 was used in combination with GA 3 , the germination percentage increased significantly. This may be attributed to the fact that nitrate reduces abscisic acid concentration in seeds which is responsible for dormancy, whereas GA 3 promotes germination (Hilhorst & Karssen 1992). The germination percentage (46.19 ± 3.59%) of seeds under control condition (soaked in distilled water) took the maximum number of days (39.11 ± 0.40) for germination (p <0.05) as compared to other treatments (Table 1).
In vitro seed germination of I. embelioides was observed after 40 days of culture both under light and dark conditions (Image 1C). ANOVA of the result revealed that the germination percentage sharply decreased in seeds kept under the dark-as compared to light-conditions ( Table 2). The germination percentage (65.56 ± 2.92%) was significantly higher (p <0.05) in seeds cultured in media containing 10 mg L -1 GA 3 under light condition. This result was in agreement to the in vitro seed germination observed in Ilex brasiliensis, I. pseudoboxus and I. theezans (Dolce et al. 2015).
Both warm and cold stratification helped in breaking dormancy of I. embelioides seeds. Seeds warm stratified (25 ± 1°C) for 30 days showed the highest germination percentage (96.67 ± 1.93%) after 40 days of incubation (24 ± 2°C). However, cold stratification was less effective compared to warm stratification and no germination was observed in seeds stratified for 40 days. Seed viability declined after 50 days of incubation irrespective of the stratification periods (Figure 1). Since embryos of the seeds require relatively high (non-cold stratifying) temperatures for growth, the species showed simple morphophysiological dormancy.
Underdeveloped embryos were observed in cold stratified seeds incubated at 24 ± 2°C for 10 to 40 days. Further, germinated seeds of I. embelioides did not exhibit a long (several-week) delay between emergence time of radicle and cotyledon and is a characteristic feature of seeds having non deep simple morphophysiological dormancy. This finding was similar to that reported in Ilex amara (Zamith & Scarano 2004), and I. nitida (Marrero 1949). The high germination of I. embelioides (>80%) when stratified at 25 ± 1°C, was similar to that observed in Ilex aquifolium, I. glabra, I. montana (Nikolaeva et al. 1985), I. opaca (Ives 1923;Barton &Thornton 1947), I. verticillata andI. vomitoria (Nikolaeva et al. 1985) and I. maximowicziana (Chien et al. 2011). Thus, warm stratification could break non deep simple morphophysiological dormancy.
The viability of the seeds declined with the storage period irrespective of how they were stored. However, seeds stored at 25°C with moist sand showed the highest viability percentage (54.72 ± 1.67%) even after 30 days. Whereas, seeds stored in airtight container For each treatment, means followed by the same letter in each column do not differ significantly at p <0.05 (Tukey test). declined their viability to 91.11 ± 1.00% after 10 days and completely lost viability after 20 days. Viability of the seeds stored in air tight polybag at 5°C declined to 41.67 ± 0.83% and 12.78 ± 0.74% after the 20 th and 30 th day, respectively. Seeds stored in moist sand at 5°C remained 100% viable after 10 days but the viability declined to 86.94 ± 1.55% and 43.05 ± 0.28% after 20 th and 30 th days of storage. The comparison of viability percentage of I. embelioides seeds subjected to different storage conditions is presented in Figure 2. The low viability of I. embelioides revealed that the seeds should be germinated as early as possible.

CONCLUSION
Under natural conditions the low seed germination and high exploitation of Ilex embelioides poses a serious threat for its very existence. The seeds of I. embelioides exhibit simple morphophysiological dormancy which means the seeds have underdeveloped embryo and physiological dormancy. Application of GA 3 and warm stratification (25 ± 1°C) of seeds for 30 days in moist sand are recommended for germination. The in vitro

J TT
seed germination experiment has demonstrated that seed cultured on MS medium with 10 mg L -1 GA 3 is also effective for mass propagation. Though viability was high in seeds stored at 25°C in moist sand, the viability declined with the storage period. This indicates that the seeds should be germinated as early as possible. The suitable protocols for mass multiplication of the species developed (Image 1D) in the present study would help not only in reducing the germination time but also to obtain large number of seedlings for reintroduction of the species in its natural habitat.