Morphological characterization of citrus cultivars in the subtropical region of Himachal Pradesh

Introduction

Citrus is one of the most important fruit crops globally, ranking third in total fruit production after banana and grapes (FAO, 2021) [8] . India, being part of its centre of origin, holds considerable genetic diversity in citrus, particularly in the North-Eastern Himalayan and Western Himalayan regions (Swingle and Reece, 1967; Singh and Chadha, 1993) [25,21] . Among citrus species, mandarin (Citrus reticulata Blanco) and sweet orange (Citrus sinensis L. Osbeck) dominate the Indian citrus industry, contributing significantly to fruit production, processing industries and farmer livelihoods. Citrus fruits are also valued for their high nutritional content, being excellent sources of vitamin C, carotenoids and bioactive antioxidants that help to reduce the risk of chronic diseases such as cardiovascular disorders, diabetes and certain cancers (Gil-Izquierdo et al., 2001; Abd-Ghafar et al., 2010) [9,1] . Botanically classified as hesperidia, citrus fruits show remarkable variability in fruit morphology, rind colour, pulp texture and seed traits, making phenotypic characterization an essential tool for cultivar identification, breeding, conservation and germplasm management (IPGRI, 1999) [10] . With the increasing demand for quality planting material and high-density orchard systems, precise varietal identification and characterization have become critical. However, the highly heterozygous nature of citrus, presence of polyembryony, frequent bud mutations and intergeneric hybridization often complicate species delimitation and cultivar identification (Talon and Gmitter, 2008) [26] . In such cases, standardized morphological descriptors provide a practical and reliable means of distinguishing closely related cultivars, especially under diverse agro-climatic conditions. Morphological characterization plays a crucial role in the assessment of genetic diversity, cultivar identification and improvement programmes. It offers a cost-effective and practical approach for evaluating phenotypic variability among citrus genotypes. Important traits such as tree habit, leaf morphology, fruit shape, rind texture, pulp characteristics and seed traits serve as reliable markers for distinguishing cultivars ( https://www.biochemjournal.com [11, 13] . Understanding morphological variation under specific environmental conditions helps growers to select suitable cultivars and enables breeders to identify promising parents for hybridization. The present investigation was conducted to characterize the variability among seven citrus cultivars, including three cultivars of mandarin (Kinnow, Daisy, W. Murcott) and four cultivars of sweet orange (Blood Red Malta, Jaffa, Mosambi, Valencia Late), under the subtropical conditions of Himachal Pradesh. The study aimed to evaluate tree, foliage, fruit and seed traits using standardized IPGRI descriptors to identify cultivar-specific characteristics and assess phenotypic diversity. The findings will aid in effective germplasm management; guide cultivar selection and support breeding programmes adapted to specific agroclimatic conditions.

Materials and Methods

Planting material and site

Morphological characterization

Morphological characterization was carried out based on 32 qualitative traits, following the Descriptors of Citrus by International Plant Genetic Resources Institute (IPGRI, 1999) [10] , now renamed as Bioversity International. Morphological data pertaining to tree, foliage, fruit and seed traits were collected during two successive growing seasons.

Tree vegetative characters

Spine density was assessed by counting the number of spines per 50 cm of shoot length on five randomly selected shoots per tree and classified as absent, low, medium or high. Spine shape was recorded as curved or straight.

Foliage characters

Vegetative life cycle was classified as evergreen, deciduous or semi-persistent by visually observing the leaf retention pattern throughout the season. Ten leaves per tree were evaluated for leaf division (simple to pentafoliate), intensity of green colour (light to dark), lamina attachment (sessile, brevipetiolate or longipetiolate) and shape (elliptic to obcordate). Leaf margins were categorized as crenate, dentate, entire or sinuate, while the shape of leaf apex ranged from attenuate to emarginate. Presence or absence of petiole wings was also recorded.

Fruit characters

Five fully mature fruits from each plant were evaluated for fruit shape (spheroid to ovoid), base (necked to concavecollared) and apex (mammiform to depressed). Fruit skin colour was recorded using a 12-point colour scale (green to red-orange). Fruit surface texture was scored as smooth, rough, papillate, pitted, bumpy or grooved. Density of oil glands was calculated by counting the number of oil glands per square centimetre on five fruit surfaces per replication and categorized as low, intermediate or high. Albedo colour and presence of areola was recorded based on visual inspection. Adherence of segment walls was determined based on the ease of segment separation and classified as weak, medium or strong. Segment wall thickness, fruit axis (solid to hollow), pulp colour (white to purple) and pulp firmness (soft to firm) were also evaluated visually.

Seed characters

Ten seeds per replication were analysed for shape (fusiform to semi-spheroid), surface texture (smooth to hairy) and colour (white to brown). Seed embryony was assessed by dissecting seeds and classifying them as monoembryonic, polyembryonic or mixed.

Results and Discussion

The morphological characterization of seven citrus cultivars was carried out to evaluate the phenotypic variability in tree, foliage, fruit and seed traits. The results presented here are based on observations recorded over two consecutive years and are discussed in relation to existing descriptors and relevant literature.

Tree vegetative characters

Among the seven citrus cultivars evaluated, low spine density was observed in Kinnow, Blood Red Malta, Jaffa, Mosambi and Valencia Late, while spines were completely absent in Daisy and W. Murcott (Table 1). No variation was recorded in spine shape among the mandarin and sweet orange cultivars. These results are in line with the earlier observations of Singh (2006) [23] and Singh et al. (2010) [22] .

Foliage characters

The citrus cultivars under study exhibited both uniformity and subtle variation in their foliage traits (Table 2). All cultivars showed an evergreen growth habit, with simple leaf division and brevipetiolate leaf lamina attachment. Petiole wings were present in all cultivars. These results are consistent with earlier findings reported by Khan et al.

(2008) [13] , Talukder et al. (2015) [27] and Kaur et al. (2016) [12] . In terms of leaf blade colour, slight variability was observed. Kinnow, Blood Red Malta, Jaffa, Mosambi and Valencia Late exhibited a dark green colour, whereas Daisy and W. Murcott had medium green foliage. Jaskani et al.

(2006) [11] reported comparable results, recording dark green leaf blades in Bitter sweet orange and Sour orange and medium green in Yuma citrange.

Leaf lamina shape exhibited moderate diversity among cultivars. W. Murcott, Blood Red Malta, Jaffa and Valencia Late were characterized by an elliptic lamina, while Daisy and Mosambi had ovate-shaped leaves. Kinnow was distinct in having a lanceolate leaf shape. Similar variation in leaf form was documented by Marboh et al. (2015) [15] in different Indian citrus genotypes and by Egbadzor et al.

(2022) [7] in a morphological assessment of citrus accessions in Ghana.

Variations were also recorded in the leaf margin and apex shape. Dentate leaf margins were observed in Kinnow, Daisy, W. Murcott and Jaffa; crenate margins were found in Blood Red Malta and sinuate leaf margins in Mosambi and Valencia Late. These patterns align with Dubey et al. (2019) [6] , who described similar margin types in Citrus maxima accessions. The leaf apex was predominantly acute across all cultivars, except for Mosambi, which exhibited an obtuse apex. Similar trends were documented by Nurzaman et al.

(2024) [17] in a phenotypic analysis of citrus genotypes.

Fruit characters

The analysis of fruit-related traits across the citrus cultivars revealed noticeable variability in several morphological characteristics, while some traits remained consistent among the cultivars. The overall fruit shape was found to be spheroid in all the cultivars except W. Murcott, where fruits were obolid (Table 3). Fruit base was reported as truncate in all the cultivars except for Daisy where it was recorded as convex in shape. The fruit apex was reported as truncate in Kinnow, Daisy, W. Murcott, Blood Red Malta and Jaffa, while rounded apices were found in Mosambi and Valencia Late. Similar findings were reported by Altaf et al. (2014) [3] and Sunaiana et al. (2018) [24] . Fruit skin colour (epicarp) varied among the cultivars. Kinnow exhibited a typical orange skin colour, Daisy and W. Murcott had dark orange colour, while light orange colour was observed in Blood Red Malta, Jaffa, Mosambi and Valencia Late. These observations correspond with the findings by Patel et al. (2016) [18] , who reported diverse skin colours in both sweet orange and mandarin groups. Surface texture exhibited moderate variation. Fruits of Kinnow, Daisy and W. Murcott exhibited smooth rind texture, whereas Blood Red Malta was characterized by a distinctly rough and uneven rind. Pitted textures were observed in Jaffa, Mosambi and Valencia Late. Dorji and Yapwattanaphun (2011) [5] similarly reported smoothskinned fruits in mandarins, while Usman et al. (2020) [28] observed smooth texture across all studied grapefruit cultivars. No significant variation was observed in oil gland density or presence of areola. All cultivars had a low density of oil glands (<40/cm 2 ) and an areola was consistently present. These findings are in agreement with those of Baswal et al.

(2015) [4] and Sunaiana et al. (2018) [24] , who reported similar traits in sweet orange cultivars. Albedo (mesocarp) colour showed slight variability. White coloured albedo was observed in all the cultivars except for W. Murcott where it was recorded as orange in colour. These results support earlier findings by Jaskani et al. (2006) [11] . Segment-related traits also varied among cultivars. Kinnow, Daisy and W. Murcott had medium adherence between segment walls, while strong adherence was seen in Blood Red Malta, Jaffa, Mosambi and Valencia Late (Table 4). Thin segment walls were present in the former group, whereas the latter cultivars had medium-thick segment walls. Fruit axis was solid in Kinnow, Daisy and Valencia Late and semi-hollow in the remaining cultivars. These findings align with previous studies by Talukder et al.

(2015) [27] , Singh et al. (2016) [20] and Raheb et al. (2018) [19] , who documented similar segment traits in citrus varieties. Pulp characteristics also showed variation. Pulp (flesh) colour was orange in Kinnow, Daisy and W. Murcott; orange-red in Blood Red Malta and yellow in Jaffa, Mosambi and Valencia Late. Pulp firmness was soft in the mandarin cultivars and intermediate in the remaining cultivars. These results align with those of Usman et al.

(2020) [28] and Zacarías-García et al. (2021) [31] , who reported comparable differences in pulp colour and firmness in different citrus cultivars. Among all the traits evaluated, fruit morphology exhibited the greatest degree of variability, underscoring its significance in differentiating citrus cultivars.

Seed characters

The citrus cultivars studied exhibited considerable diversity in seed morphological traits. Seed shape varied across the cultivars: Kinnow and W. Murcott had clavate shaped seeds, Daisy and Valencia Late were characterized by semi-deltoid seeds, while Blood Red Malta, Jaffa and Mosambi had ovoid seeds (Table 5). These differences highlight the morphological diversity present among the evaluated citrus cultivars. Seed surface texture also varied among cultivars. A smooth surface was observed in Kinnow, Daisy and W. Murcott, whereas the remaining cultivars had a wrinkled seed surface. These results are in agreement with those reported by Malik et al. (2012) [14] and Ahmed et al. (2015) [2] . In contrast to shape and surface texture, no variation was observed in seed colour and embryony among the studied cultivars. All cultivars consistently had cream-coloured seeds that were polyembryonic in nature. This aligns with genomic evidence from Wang et al. (2022) [29] , which highlights widespread nucellar embryony in cultivated citrus. Woo et al. (2019) [30] and Martínez-Ochoa et al.

(2022) [16] also documented consistent polyembryony in sweet orange accessions.

Conclusion

The study revealed considerable morphological variability among the evaluated citrus cultivars, particularly in traits related to leaves, fruits and seeds, whereas certain vegetative characteristics remained largely uniform. These phenotypic variations hold practical significance for cultivar identification, trait-based selection and crop improvement programmes. Morphological traits, being simple and fieldadaptable, provide an effective tool for preliminary screening and genetic resource management. The diversity observed, especially in commercially relevant traits such as fruit skin and pulp colour, can be utilised to select potential donors for future citrus breeding initiatives.

Acknowledgement

AcknowledgementThe authors express their sincere gratitude to the Department of Fruit Science, College of Horticulture and Forestry, Neri, Hamirpur, Himachal Pradesh, India, for providing the necessary facilities, continuous support and valuable guidance throughout the duration of this research.

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