Variation in seed morphology and selected oil parameters of neem ( Azadirachta indica A. Juss.) from different agroclimatic zones in Tamil Nadu, India

SUMMARY: Tamil Nadu, in southern India, has the second-largest number of neem trees in the country. The oil from the seeds has high economic significance for cottage industries in the region. This paper examines 28 Candidate Plus Trees (CPTs) selected from six agroclimatic zones in Tamil Nadu which exhibit exceptional traits such as superior growth and other desirable characteristics. We aimed to understand seed morphology variations and physicochemical properties in the oil across different regions. Significant differences were observed for morphometric traits. Fruit production correlated negatively with rainfall. 100-seed kernel weight and seed length correlated with oil percentage. Rainfall influenced seed breadth and pericarp weight. Clustering using morphological characters did not group genotypes from the same region; while soil type could distinguish them. Correlation helped us determine the prominent features which influence the traits of interest, which can be useful for b reeding programs, cultivation practices, and th e development of n eem-based products in Tamil Nadu and beyond.


INTRODUCTION
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.

Collection of fruits
An extensive survey was carried out during 2019-20 in different agroclimatic regions of Tamil Nadu to select superior phenotypes of neem.Tamil Nadu has wide variations in its agro climatic zones (ACZ).The state is divided into seven ACZs.The present study covered six zones, leaving behind the Hilly region (Nilgiris), where neem trees are not present (FSI, 2019).The agroclimatic zones with precipitation and soil details are shown in Table 1.The details of selected Candidate Plus Trees (CPTs) concerning fruit yield, growth superiority, pest and disease incidence are presented in Table 2.The data on height, girth at breast height (GBH), clear bole height (CBH) and crown diameter were recorded and the seeding behavior of the selected CPTs was scored on a scale of 1 to 5 (1-Poor fruiting and 5 -Heavy fruiting).Fully mature, yellowish-green fruits were collected from the CPTs.Within 24 hours of collection, the fruits were de-pulped and their seeds were washed thoroughly with tap water to remove pulp, dirt and impurities.The seeds were shade-dried and stored under ambient conditions.All experiments were conducted in a completely randomized design with four replicates.

Measurement of seed morphometric characters
Neem seeds (100 in four replicates -25 in each) were spread on the glass plate of a macro viewer and images were captured.Using Leica Q Win, the length (cm) and breadth (cm) of the seeds were recorded.The 100-seed weight (g), 100-seed kernel weight (g) and pericarp weight (g) of the seeds were estimated using an electronic balance and pericarp percentage was calculated from the recorded values.The moisture content of the neem seeds was estimated as per ASAE (1998).

Oil extraction and characterization
Twenty grams of seed kernel powder were used for oil extraction.The oil content was extracted using the standardized Soxhlet method.Petroleum ether was used as solvent.

Physical properties
Oil color was determined visually and the odor was determined by the volatilized smell.The pH of the oil samples was analyzed using pH indicator strips (Merck, Germany).The refractive index (RI) of the oil was estimated using the standard method of AOAC (2007) and quantified using a pen Refractometer (Atago, Japan) with a resolution and accuracy value of ± 0.1 and ± 0.2%, respectively at 10-60 °C.The specific viscosity of the oil was measured according to the standard method ASTM (2003) at 30 °C using the Engler Viscometer and expressed in Degrees Engler (°Engler).

Chemical properties
Acid value.The acid value was determined according to the method described by AOAC (2007) and expressed as the KOH (in mg) necessary to neutralize the free fatty acids contained in 1 g of oil.
Saponification value.A measurement of the fatty acid chain length in oils was determined by standard procedures (AOAC, 2007) and expressed in milligrams KOH absorbed per gram of oil.
Iodine value.The iodine value was estimated by Oomah et al. (2000).
Peroxide value.The peroxide value was determined by Cox and Pearson (1962) and expressed in meq O 2 •kg -1 of oil.
Specific gravity.Specific gravity was measured following the standard method of AOCS (1997).

Statistical analysis
A statistical analysis was conducted using SPSS (v.20).Analysis of variance followed by post-hoc (Duncan's Multiple Range Test (DMRT)) was performed at a 5% significance level.Correlation analyses were employed to find the relationship between the meteorological data and seed morphology with the oil contents.The dendrogram was constructed by the Ward method of cluster analysis on the Average distances between zones.To determine the robustness of the dendrogram, the data was bootstrapped with 1000 replicates.

Variations in plus trees and morphometric characteristics of the seeds
The tree height of the individual trees varied from 8.98 m in IFGTB AI 29 to 17.22 m in IFGTB AI 32; whereas the highest GBH was recorded in IFGTB AI 28 (186 cm) and the lowest value in IFGTB AI 29 (110 cm) (Table 2).Variations were also observed among the different ACZs.The GBH and seeding behavior among the ACZs showed significant (P < 0.05) variations.The highest GBH was recorded in ACZ 5 (186 cm), while ACZ 3 recorded the lowest (125 cm) values.Seeding behavior was highest in ACZ 3 and lowest in ACZs 1 and 6.A strong (0.531) negative correlation was observed between seeding behavior and total annual rainfall (data not shown), indicating the influence of yearly rainfall on seeding behavior.Crown diameter, however, did not show any relation to seeding behavior.
There were significant (P < 0.05) variations among the ACZs as well.ACZ5 recorded high values for seed length, 100-seed and kernel weight and oil percentage.However, ACZ 4 showed high values for breadth, pericarp weight and pericarp percentage (Table 4), suggesting fruits with thicker pericarps.
The correlation matrix of seed characteristics, oil content and rainfall are given in Table 5. Seed morphometric characteristics such as 100-seed weight (r = 0.959), 100-kernel weight (r = 0.848) and seed length (r = 0.874) recorded strong positive correlation with oil percentage of neem seeds.However, the correlation between seed breadth and oil percentage was negligible (r = 0.08).Rainfall influenced seed breadth (r = 0.311) and pericarp weight (r = 0.308).The total minimum and maximum rainfall influenced pericarp percentage (Table 5).

Physical properties
The physical properties of neem seed oil across the plus trees (Table 6) and zone-wise (Table 7) reveal significant differences only in specific gravity.No significant differences were observed among trees within the same zone.The highest specific gravity was observed in oil from ACZ4 (0.87) and the lowest in ACZ 3 (0.72).The viscosity of the neem oil in zones ranged from 44.8 to 53.59 mm 2 •s -1, with values for individual trees ranging between 44.54 mm 2 •s -1 and 58.88 mm 2 •s -1 .The oil's refractive index was the highest in IFGTB AI 4 (1.67) and the lowest in IFGTB AI 2 (1.45).The average pH in the seed oil was 4.64 and the maximum was recorded for IFGTB AI 34 (5.5) and minimum for IFGTB AI 16 (4.05)(Table 6).

Chemical properties
The chemical properties of neem seed oil are shown in Table 7. Except for peroxide values, all other chemical properties were significantly different (P < 0.05) across zones and among chemical properties (Table 7).Zone ACZ 2 recorded high values for both saponification and iodine values.ACZ 6, 5 and 3 recorded high values for saponification, acid and peroxide values, respectively.The highest saponification value in the oil was obtained for IFGTB AI 37 (319.54mg KOH•g -1 ), followed by IFGTB AI 10, IFGTB AI 29 and IFGTB AI 41 (312.98 mg KOH•g -1 ), and the lowest was detected in IFGTB AI 12 (122.96mg KOH•g -1 ) (Table 6).The acid value of the oil was found to be at its highest in IFGTB AI 41 (38.01 mg KOH•g -1 ) and its lowest in IFGTB AI 12 (18.99mg KOH•g -1 ).The highest iodine value in the oil was found for IFGTB AI 41 (70.3 gI•100 g -1 ), followed by IFGTB AI 34 (67.77g•ml - ), with the minimum value recorded for IFGTB AI 13 (24.59gI•100 g -1 ) (Table 6).
Clustering revealed that neem from ACZ 3 was distinct from the rest of the zones.In the second clade, neem from ACZ 5 was clustered separately.ACZs 1 and 6 were grouped together.Likewise, ACZs 2 and 4 were grouped together.

DISCUSSION
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 Depulped Neem Seed Oil (Reaffirmed in 2018) prescribes a specific gravity range of 0.908-0.934for 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.

CONCLUSIONS
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.

Figure 1 .
Figure 1.Dendrogram of neem from different Agro-climatic Zones (ACZ) studied based on Ward method using Average linkage.

Table 1 .
Agroclimatic zones (ACZ) with the number of trees selected in parenthesis, along with precipitation and soil details

Table 2 .
Variation in tree morphometric traits in the selected Candidate Plus Trees (CPTs) of Azadirachta indica.

Table 3 .
Variations in the morphological characteristics of Neem seeds collected from different parts of Tamil Nadu *Values are means of four replicates.IFGTB AI: Institute of Forest Genetics and Tree Breeding Azadirachta indica.

Table 5 .
Correlation studies of seed parameters and oil content in Azadiracta indica with precipitation

Table 6 .
Variations in physical and chemical properties of Neem oil extracted from seeds collected from different parts of Tamil Nadu *Values are means of four replicates.IFGTB AI: Institute of Forest Genetics and Tree Breeding Azadirachta indica.

Table 7 .
Physical and chemical properties of neem seed oil from different agro-climatic zones (ACZ).Means compared using DMRT.