Azadirachta indica A. Juss., (Neem; Family: Meliaceae), an evergreen, multipurpose tree which is native to India, grows in the arid, semi-arid, and tropical conditions in Pakistan, Bangladesh, Srilanka, Malaysia, Indonesia, Thailand, the Middle East, Sudan and Niger (Kaushik et al., 2007). The species shows wide adaptability. It is seen as an avenue, ornamental agroforestry or roadside tree. It grows in clay, saline, alkaline, dry, stony, shallow soils, including high calcareous soil (Pattnaik et al., 2006, Atabani et al., 2013), and tolerates high temperatures, low rainfall, long spells of drought and salinity. For centuries, it has been used in traditional medicine. Various parts are used in the Ayurvedic and Unani systems of medicine. It is estimated that ~ 25 million neem trees exist in India, the highest recorded from Uttar Pradesh, followed closely by Tamil Nadu, Madhya Pradesh, Andhra Pradesh, and Karnataka. A fully grown tree produces about 50 kg fruits annually. The productivity of neem oil mainly varies from 2 to 4 t/ha/yr (Kaushik et al., 2007).

Neem Seed Kernel Extract (NSKE) is widely used in agriculture. About 20-30% of the seed weight constitutes oil - the kernels contain 40-50% of an acrid green to brown oil (Atabani et al., 2013). The oil comprises fatty acids, mainly oleic acid (13.50- 26.76%), palmitic acid (6.88-11.99%), linoleic acid (7.32-11.17%), stearic acid (4.29-13.08%), and arachidic acid (0.53-1.27%) (Adigwe et al., 2022). Azadirachtin, salannin, and other limonoids are also present in small quantities. Neem oil, being one of the least toxic to humans and beneficial organisms, is very promising in the control of numerous pests.

Kaushik et al. (2007) and Jessinta et al. (2014), provide information on the variation in tree morphology, vegetative and reproductive phenology, and seed morphology of the neem species across different geographic locations. However, there is a lack of information specifically related to agroclimatic zones. Tamil Nadu is the second in India’s highest number of neem trees, so we felt it imperative to understand the variations across agro-climate zones. This study is significant considering the state’s high number of neem trees and their relative abundance (12%), as reported by the Trees Outside Forests (TOF) inventory (FSI, 2019).

Hence, this paper discusses variations in seed morphology and seed oil properties in neem from various agroclimatic regions of Tamil Nadu. It would also enable us to predict site-source matched provenances for future planting.

Cataloguing seed morphological features from natural populations is considered the first step in understanding the genetic variability of a species. Multiple factors could induce and maintain variation in seed features. Large seeds are favored as they produce large and vigorous seedlings. On the contrary, smaller seeds may have a better selection advantage due to broader and more effective dispersal (Eriksson 1999). Fruit and seed characteristics, namely weight, length, width, diameter, yield and oil content, are reported highly variable both within and among the provenances of neem (Kundu and Tigerstadt, 1998; Jindal et al., 1999). The present study also revealed significant variation among individual trees (CPTs) and among agroclimatic zones. Kundu and Tigerstadt (1997) reported distinct clustering of neem provenances based on rainfall regions. A strong negative correlation was observed in the present study between seeding behavior and total annual rainfall, in line with earlier reports. Thus, it could be concluded that rainfall plays a crucial role in seed-bearing.

Seeds are influenced by various factors, such as geographic area, climate, genetic variability, agronomic conditions, plant morphology and physiology, collection and storage of plant material (Fernandes et al., 2019). Though our reports are consistent with reports on variability in seed length in neem seeds collected from five provenances in northern and western India (Kaura et al., 1998), the 100-seed weight ranged from 10.14 to 36.38 g, while they report 0.8 to 3.5 g, indicating smaller seeds. Variation in seed parameters such as seed diameter, seed length, kernel-to-seed ratio, 100-kernel weight and 100-seed weight have also been reported by Gupta et al. (2012) in different provenances in Gujarat. Kumaran et al. (1993) reported high heritability for seed length, seed oil content, and 100-seed weight. These parameters could be a robust selection index for neem.

We also obtained significant correlations between oil content and 100-seed and kernel weight in accordance with their findings. Neem seeds collected from different locations in Tamil Nadu showed a positive correlation between oil content and the number of hours of sunshine (Sridharan et al., 1998). As presented in Table 4, rainfall influenced seed breadth, pericarp weight and percentage, suggesting that rounder fruits with thicker pericarps could be observed in high rainfall areas. Seed and kernel weight, which positively correlated with oil content, can be considered promising traits for the early selection of seed sources. Similar results have been reported for other tree-borne oil seeds (Kaura et al. 1998).

Seed oil content varies among tree-borne oil seeds (Vollmann et al. 2007). Oil yield is also affected by tree age, seed extraction method, seed storage, and environmental factors. This variation in the present study and other seed morphological attributes presents us with a viable selection alternative from base seed material at a very early stage. This could be useful for the improvement of programs, especially considering that the neem is commercially important for its oil and azadirachtin contents. We recorded 14 to 60% oil content in neem seed kernels, similar to Kaura et al., (1998) and Tomar et al. (2011), who report the influence of agroclimatic zones on oil content.

The present study recorded the highest oil percentage from ACZ 5, comprising alluvial soil, which is rich in minerals, especially potash. This may have contributed to its high oil content, as indicated by Devaranavadagi et al. (2003), who reported oil content variation due to climatic and site conditions. Sidhu et al. (2003) also reported low oil contents in neem from arid, saline and coastal regions.

The Bureau of Indian Standards (BIS) IS 4765: 1975 Specification for Neem Kernel Oil and De-pulped Neem Seed Oil (Reaffirmed in 2018) prescribes a specific gravity range of 0.908-0.934 for neem oils. None of the samples fell within this range. The BIS also defined ranges for saponification value (188-205 mg KOH·g^-1), iodine value (65-80 gI·100 g^-1) and acid value (15 mg KOH·g^-1).

Acid value is a relative measure of rancidity as free fatty acids, while iodine value defines the drying quality of the oil. The low iodine values obtained for the samples studied represents the fewer unsaturated bonds, indicating that the low tendency of the oil to undergo oxidative rancidity. Accordingly, the peroxide value, an indication of the rate of rancidity was also low (Table 7). The presence of water or moisture contributes to hydrolysis, thus leading to higher acid values, and reducing the storage capacity of the oil (Do et al., 2022).

The saponification value, a measure of the average molecular weight of all the fatty acids in the sample in triglycerides, varied from 190.35 to 299.6 mg/KOH. Hussein et al. (2021) also reported a wide range of variation in neem. A higher acid value increases saponification value (Hussein et al., 2021), thus increasing the possible utilization of the oil in soaps and cosmetics. The proportion of each fatty acid in the oil may vary from tree to tree because of genetic make-up. A high saponification value implies the potential tendency to soap formation, and long-stored degraded oils are good for soaps and toiletry product productions (Hussein et al., 2021). The prevalence of a wide variation in the neem accessions collected from different agroclimatic zones helped us classify the oil’s utility prospects from the different agro-climatic zones. Genotypes with higher saponification values could be recommended for the cosmetic industry.

Morphometric techniques are valuable tools for exploring population differentiation, allowing more rigorous comparisons within a genus (Kolawole et al. 2016). Morphological characterization reveals diversity between germplasm. The clustering of zones based on seed morphology and oil content revealed that soil played a significant role in grouping the accessions. The saline coastal (ACZ 3) and rich alluvial (ACZ 5) zones remained distinct. However, it is to be noted that distinction based on morphological characteristics may not cluster genotypes from the same region within the same group (Figure 1). However, Kolawole et al. (2021) suggested phenetic dendrograms as the first step to grouping variables, dissecting the prominent features which influence the traits of interest, followed by a rigorous selection.

Understanding the variations in seed morphology and seed oil properties across agroclimatic zones allows for a comprehensive understanding of the species’ adaptability and response to different climatic conditions. This knowledge can aid in selecting appropriate neem provenances for future planting in specific agroclimatic regions. Variations in seed morphology and seed oil properties have implications for various applications of neem products. Neem oil, derived from the seeds, is widely used in agriculture, medicine, cosmetics, and other industries. The quality and composition of neem oil is observed to vary based on geographical factors, which in turn affects its efficacy and suitability for different purposes.

Studying the seed morphology and seed oil properties across various agroclimatic regions in Tamil Nadu provided insights into the geographic variations within the neem species. This information contributes to the development of site-source matched provenances, helping in the selection of neem trees that are well-suited to specific agroclimatic conditions.

Cottage industries in Tamil Nadu extensively utilize neem for the production of various products. Neem oil, extracted from neem seeds, is a primary ingredient in the manufacturing of soaps, shampoos, hair oils, and other personal care items. Neem-based products from Tamil Nadu’s cottage industries have the potential for export, contributing to the state’s economy. With a wide distribution of the species in the state, assessing the extent of variability becomes pertinent. This paves the way for developing a defined tree program by targeting oil. Soil type and rainfall influenced oil content. Of the twenty-eight superior trees identified from six agro-climatic zones, the oil content was the highest in those growing in alluvial soil. Fruit yield was also dependent on rainfall. Information on the influence of environmental variables is crucial to identify high oil-yielding populations. This would enable the selection of genotypes for developing improved and adapted cultivars. Superior genotypes from these selections can also be used for establishing large scale plantations.