Effects of gibberellic acid and chitosan on seed germination and Morpho-physiological parameters of sandalwood (Santalum Album L.)

Introduction

Indian Sandalwood (Santalum album L.) stands as a highly coveted species within Indian forestry, renowned globally for its premium commercial timber, valuable heartwood and aromatic essential oil. The species demonstrates a broad geographic range occurring naturally across latitudes from 30°N to 40°S. Within India, indigenous populations are primarily concentrated in Kerala and Andhra Pradesh; however, fragmented populations have been documented across a diverse array of states including Bihar, Gujarat, Haryana, Maharashtra, Madhya Pradesh, Odisha, Punjab, Rajasthan, Uttar Pradesh, and West Bengal, with more recent sightings even reported in states of Himachal Pradesh and Assam. As a moderately sized evergreen, Sandalwood typically reaches a height of 12 to 15 meters with a girth ranging from 1 to 2.4 meters, characterized by both erect and slenderly drooping branches (Sen Sarma, 1982) [14] . This adaptable species flourishes at elevation ranging from sea level up to altitudes of 1,800 meters. It particularly favors cool climates with extended dry periods and moderate annual rainfall of 600 to 1,600 millimeters (Troup, 1921) [18] . Indian Sandalwood exhibits an early reproductive cycle, often initiating flowering between two to three years of age, though specific flowering and fruiting seasons can vary. The tree produces succulent, globose to elongated drupe fruits that ripens to deep purple black. Each fruit encases a spherical seed enclosed within a moderately hard brown endocarp, though the fruit's overall shape can range from globose and ovate to elongated with tapering ends. Propagation predominantly relies on seeds which are yielded either once or twice annually, typically between June and September or November and February. Sandalwood seeds demonstrate a remarkably prolonged period of seed dormancy (Doran et al. , 2005) [6] . Consequently, the germination process is protracted, requiring anywhere from 4 to 12 weeks to complete (Srinivasan et al. , 1992; Srimathi et al. , 1995) [16,17] . This intense dormancy is multifaceted; it is primarily driven by a seed coat that is impermeable to both water and oxygen, alongside other factors such as mechanically resistant seed coats and rudimentary or physiologically immature embryos (Das and Tah, 2013) [5] . Seed dormancy is the most limiting factor for seed germination and thus it causes prolongation in seed germination. Sandalwood has emerged as a highly adaptable botanical asset, demonstrating significant utility across the agricultural, industrial, and medical sectors. The species acts as a rich biological reservoir of phytochemicals that are increasingly sought after by the modern cosmetics and pharmaceutical sectors. As natural reserves deplete amidst surging global demand, the economic value of sandalwood has escalated dramatically. This lucrative market environment has catalyzed a unified push toward commercial cultivation, drawing collaborative interest from farmers, policymakers and industry executives. To successfully sustain these widespread commercial initiatives, it is imperative to optimize propagation techniques. Specifically, there is a critical need to evaluate various chemical pre-sowing treatments to maximize seed germination rates and ensure the production of high vigour seedlings.

Materials and Methods

The experiment was carried out in the year 2025-26 at Research Farm of Agroforestry, All India Coordinated Research Project (AICRP) on Agroforestry and Agricultural Botany section, College of Agriculture, Nagpur. The field experiment was conducted using Randomized Block Design (RBD) with three replications and eight treatments. The matured uniform fruits of sandalwood were collected from the mother plants situated at AICRP on Agroforestry, Nagpur. To extract the seeds, the collected fruits were initially soaked in water and rubbed against a hard surface to macerate and remove the pulp. Following the extraction, seeds were dried in the shade. Prior to sowing, the seeds were subjected to a 24-hour soaking period in the given eight treatments consisting of T1 -Control, T2 -Water soaking, T3 -GA3 @ 100 ppm, T4 -GA3 @ 500 ppm, T5 -GA3 @ 1000 ppm, T6 -Chitosan @ 100 ppm, T7 -Chitosan @ 500 ppm, T8 -Chitosan @ 1000 ppm. To mitigate the risk of fungal attack, all seeds were treated with fungicide. For each treatment, 50 seeds were sown by dibbling method in polythene bags of size 6 x 8 inches filled with a mixture of FYM, soil and cocopeat in 1:2:1. Red Duranta (Duranta erecta L.) was co-planted as a primary host plant. The polythene bags were maintained in shaded greenhouse environment. Observations were recorded on five random plants at 50, 100 and 150 days after sowing (DAS) in each treatment. The observations such as germination percentage, plant height, number of leaves, collar diameter, shoot length, root length, fresh weight of root, fresh weight of shoot, dry weight of root, dry weight of shoot and leaf area per plant, leaf area index (LAI), crop growth rate, net assimilation rate were recorded. The plant height was measured from the base to the tip of the observational plant, measured in centimeter.

The number of leaves was counted on a visual basis. The collar diameter was measured with the help of digital vernier caliper from freshly collected seedlings and recorded in millimeters. The length of the shoot was measured from the collar region to the apex region while the root length was measured from the collar region of root to the root tip and expressed in centimeters. The fresh and dry weight of both root and shoot was recorded and expressed in grams. Leaf area per plant was measured using leaf area meter and expressed in cm 2 . Leaf area index was obtained by measuring a leaf area sample plant and dividing it by the average area occupied by the plant. The crop growth rate was calculated by using the formula suggested by Watson (1958) [20] . and expressed as mg cm -2 day -1 . The net assimilation rate was calculated by using the formula suggested by Radford (1967) [13] . and expressed as mg cm -2 day -1 . The germination percentage was calculated as: Germination (%)= Number of seeds germinated /Total number of seeds sown × 100 All the experimental data were subjected to analysis of variance to determine the significance of differences, following the methodology suggested by Panse and Sukhatme (1954) [10] .

Results and discussion

Effect of gibberellic acid and chitosan on seed germination of sandalwood

The effect of the pre-treatment of gibberellic acid and chitosan has been represented in Table 1. Significant variation was observed across the chemical treatments, characterized by a progressive temporal increase in the germination percentage from 50 to 150 DAS. The data revealed that treatment T5 (GA3 @ 1000 ppm) recorded the significantly highest germination percentage of 32.00%, followed closely by treatment T8 (Chitosan @ 1000 ppm) at 27.33% and T4 (GA3 @ 500 ppm) at 25.33% at 50 DAS. T8 was found to be at par with T5. As the seedlings progressed to 100 DAS, treatment T5 maintained strict physiological dominance, peaking at 42.67%. Treatment T4 reached 37.33% which was at par with T5, narrowly outperforming T8, which recorded 36.67%. By the final observational stage at 150 DAS, the maximum germination percentage was sustained by treatment T5 at 43.33% again followed by T4 at 38.67% and T8 at 37.33%. T4 maintained the statistical parity with T5. The progressive improvement observed following gibberellic acid applications demonstrates its biochemical capacity to antagonize abscisic acid (ABA) signaling. This effectively shifts the hormonal balance from a state of quiescence to active metabolic growth (Shu et al. , 2016 [15] , Vishal & Kumar, 2018) [19] . As reported by Berry et al. (2022) [4] , seeds treated with high concentrations of GA3 (1000 ppm) recorded a maximum germination capacity in sandalwood, proving its efficacy in overcoming non-deep morphophysiological dormancy. Polaiah et al. (2020) [11] . also mentioned comparable results where the highest germination percentage was observed in seed treated with GA3 @ 1000 ppm for Santalum album L. Chitosan pretreatment also demonstrated substantial efficacy in breaking dormancy. Ali et al. (2025) [2] revealed that chitosan pretreatment significantly enhanced the germination. The robust performance of Chitosan is corroborated by Liu et al. (2022) [8] . and Malerba & Cerana (2016) [9] , who highlighted chitosan's dual-action mechanism as a biopolymer that promotes controlled hydration while upregulating metabolic mobilization in recalcitrant woody seeds.

Effect of gibberellic acid and chitosan on morphological parameters of sandalwood

The effect of different concentrations of chitosan on the morphological characters such as plant height, number of leaves, collar diameter, shoot length, root length, fresh and dry weight of both root and shoot was assessed and depicted in Tables 1, 2 and 3. The pre-treatment using both gibberellic acid and chitosan has significantly enhanced the plant height, number of leaves and collar diameter of seedlings. The data recorded at 50, 100, and 150 DAS revealed a consistent upward trajectory. At 50 DAS, T5 (GA3 @ 1000 ppm) recorded the highest plant height at 15.43 cm, number of leaves at 10.67 and collar diameter measuring 2.46 mm at 50 DAS. This was followed by T4 (GA3 @ 500 ppm) and T8 (Chitosan @ 1000 ppm). The plant heights further expanded to 17.00 cm, with number of leaves increasing to 12.67 and collar diameter to 3.13 mm at 100 DAS. This was narrowly followed by T4 and T8 while T4 was found to be statistically at par with T5. At the final stage of 150 DAS, T5 achieved an impressive peak height of 18.40 cm, with highest number of leaves at 19.33 and collar diameter at 3.18 mm followed by T4 and T8. T4 maintained statistical parity with T5. Similar observations were made by Polaiah et al. (2020) [11] . on Sandalwood regarding plant height, number of leaves and collar diameter. Also, results from chitosan treatments regarding plant height and number of leaves are in parallel with conclusions revealed by Qahraman (2026) [12] . Additionally, the structural benefits of chitosan treatments on collar diameter align with the extensive morphological studies conducted by Aleksandrowicz-Trzcińska et al. (2015) [1] . on Scots Pine (Pinus sylvestris L.). T5 (GA3 @ 1000 ppm) produced a superior shoot length of 12.78 cm and root length of 8.45 cm of seedling at 50 DAS followed by T4 (GA3 @ 500 ppm) and T8 (Chitosan @ 1000 ppm) both functioning at par with T5. As the seedlings progress to 100 DAS, T5 displayed an impressive surge, reaching 14.57 cm shoot length and 9.43 cm root length, followed by T4 and T8. At 150 DAS, the highest shoot and root length was found in treatment T5 gaining 14.79 cm and 9.58 cm respectively. This was followed by T4 and T8 both functioning at par with T5. Polaiah et.al. (2020) [11] . revealed that GA3 priming significantly maximize shoot and root length in sandalwood. Similar results regarding efficacy of chitosan on shoot and root length were reported by Zulkadir (2022) [21] . on Zea mays L. Regarding the fresh and dry weights of shoots and roots, treatment T5 (GA3 @ 1000 ppm) consistently exhibited the maximum biomass accumulation across all observational stages. At 50 DAS, T5 recorded the significantly highest fresh shoot and root weights of 0.25 g and 0.10 g, respectively, as well as the highest dry shoot and root weights of 0.06 g and 0.06 g, respectively. This superior performance was closely followed by treatments T4 (GA3 @ 500 ppm) and T8 (Chitosan @ 1000 ppm). The growth trend continued at 100 DAS, with T5 surging to maximum fresh weights of 0.47 g (shoot) and 0.13 g (root), along with peak dry weights of 0.11 g (shoot) and 0.07 g (root), which were the highest compared to the control. By 150 DAS, T5 maintained its physiological dominance, achieving peak fresh weights of 0.50 g (shoot) and 0.21 g (root) and maximum dry weights of 0.17 g (shoot) and 0.09 g (root). At this final stage, T4 demonstrated a superior root fresh weight compared to T8, while both T8 and T4 identically achieved dry root weights of 0.05 g, 0.06 g, and 0.07 g at the 50, 100, and 150 DAS stages, respectively. These findings align closely with Polaiah et al. (2020) [11] , who reported that GA3 seed priming significantly maximizes the fresh and dry weights of shoots and roots in sandalwood. Similarly, Zulkadir (2022) [21] . demonstrated that chitosan acts effectively to significantly increase both fresh and dry biomass in Zea mays L.

Effect of gibberellic acid and chitosan on physiological parameters of sandalwood

The effect of the pre-treatment of gibberellic acid and chitosan has been represented in Tables 3 and4. Significant variation was observed across the chemical treatments, characterized by a progressive temporal increase from 50 to 150 DAS. T5 (GA3 @ 1000 ppm) consistently maintained a statistically superior leaf area and leaf area index achieving 5.49 cm² leaf area with 0.28 LAI at 50 DAS as compared to T1 (Control). This was followed by T8 (Chitosan @ 1000 ppm) and T4 (GA3 @ 500 ppm) both functioning statistically at par with the superior treatment T5. T5 maintained its physiological dominance at 100 DAS by securing highest leaf area of 8.92 cm² and LAI of 0.45. This was followed by T4 and T8. At 150 DAS, T5 achieved 9.69 cm² leaf area and 0.49 LAI which is highest among all treatments. Arun et al. (2020) [3] . reported similar observations in cowpea (Vigna unguiculata L. Walp) related to leaf area whereas seed priming with chitosan similarly enhanced the leaf architecture as reported by Godase et al. (2023) [7] . Evaluated over distinct physiological intervals of 50-100 DAS and 100-150 DAS, T5 (GA3 @ 1000 ppm) maintained absolute dominance. Between 50 and 100 DAS, T5 recorded 0.057 mg cm -² day -1 and 0.159 mg cm -² day -1 of CGR and NAR respectively, which is significantly higher as compared to control. This was followed by T8 (Chitosan @ 1000 ppm) which achieved a CGR of 0.041 mg cm -² day -1 and NAR of 0.143 mg cm -² day -1 across the respective intervals, narrowly outperforming T4 (GA3 @ 500 ppm) which achieved 0.035 mg cm -² day -1 CGR and 0.134 mg cm - ² day -1 NAR at respective intervals. The treatment T5 maintained a physiological dominance, escalating CGR to 0.085 mg cm -² day -1 and NAR to 0.178 mg cm -² day -1 in the final 100-150 DAS period as compared to control. T4 (GA3 @ 500 ppm) recorded highly competitive rates of 0.163 mg cm -² day -1 NAR and CGR of 0.052 mg cm -² day -1 , slightly followed by T8 (Chitosan @ 1000 ppm) achieved 0.157 mg cm -² day -1 NAR and 0.056 mg cm -² day -1 CGR in the respective intervals. Arun et al. (2020) [3] . reported similar results regarding crop growth rate in cowpea (Vigna unguiculata L. Walp). Godase et al. (2023) [7] . reported similar results on chitosan regarding crop growth rate in lablab bean (Lablab purpureus L. Sweet).

Conclusion

The present investigation underscores the profound impact of pre-sowing chemical treatments on the germination, growth, and overall developmental potential of sandalwood seedlings. While the untreated seeds are heavily restricted by innate dormancy, the exogenous application of gibberellic acid and chitosan effectively overcomes these inherent physiological barriers. Treatment T5 (GA3 @ 1000 ppm) significantly delivered superior performance across all evaluated morpho-physiological parameters under nursery conditions, establishing it as a highly viable protocol for maximizing propagation success in Sandalwood. Furthermore, chitosan maintained robust secondary performance, serving as an effective bio-stimulant with significant potential for improving germination speed and early seedling vigour.

Acknowledgement

AcknowledgmentsI want to express my sincere appreciation to all individuals who supported and encouraged me to make this research possible. I am incredibly grateful to my research guide for the valuable guidance and support during my research. I am also thankful to Principal scientist and Head of All India Coordinated Research Project (AICRP) on Agroforestry, College of Agriculture, Nagpur and Agricultural Botany Section, College of Agriculture, Nagpur for providing me the facilities and resources to conduct this research work.

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