Morphometric and Biochemical Analysis with Seed Protein Profiling of Passiflora Species Found in the Northeastern Himalayan Region of India

Abstract

Passion fruit is an underutilised fruit in Northeastern India, known for its unique flavour and health benefits. This study analysed 15 genotypes (P1 to P15) to explore their morphological and biochemical traits related to fruit quality and yield. P. quadrangularis L. (P15) exhibited maximum flower length, fruit size, weight, juice content, shelf-life, and yield. P. edulis f. flavicarpa (P3, P5, and P2) had the highest seed count per fruit and antioxidant activity, along with greater chlorophyll and anthocyanin levels. Passiflora edulis Sims (P8 and P11) showed superior total soluble solids, carotenoids, and vitamin C. The study found that fruit shelf life positively correlated with seed weight, while the number of fruits per vine negatively correlated with seed traits and peel weight. Additionally, certain traits, such as total carotenoids, had strong positive correlations with reducing sugar and flavonoids. Principal component analysis revealed distinct trait relationships, particularly for genotypes P7 and P10. SDS-PAGE protein profiling indicated a significant distance between P3 and P14, emphasising genetic diversity. In conclusion, this research highlights the diverse morphological and biochemical characteristics of passion fruit genotypes, paving the way for the region’s improved fruit quality, yield, and breeding strategies.

1. Introduction

Passion fruit is a significant yet underexploited fruit abundantly found in the northeast region of India [1]. It is a perennial, vigorous, climbing, woody vine that produces round or oval-shaped fruits [2]. Fruits have a tough, smooth, waxy, dark purple/yellow coloured rind with faint, fine white specks [3]. The fruit contains orange-coloured pulpy juice with many small, hard, dark brown to black pitted seeds.

Passion fruit, belonging to the family Passifloraceae, encompasses 16 genera and over 700 species [4], with the genus Passiflora accounting for approximately 520 species primarily distributed across the Neotropics and Africa [5]. This diverse genus exhibits significant morphological and physicochemical variations within and between species [6,7,8]. For instance, Passiflora edulis (purple type) typically displays greater fruit length, width, and weight, while Passiflora edulis f. flavicarpa (yellow type) yields higher juice weight [9]. Fruit shapes range from round to ovoid [10], and species such as Passiflora incarnata and Passiflora coccinea differ in filament colour [11]. Variations are also evident in pulp, peel, and seed characteristics [12,13], with unique seed protein profiles, as demonstrated by distinct molecular weight bands in Passiflora foetida and Passiflora edulis via SDS-PAGE analysis [14]. The northeastern region of India, particularly the Himalayan states, hosts a rich diversity of underexploited passion fruit species, including Passiflora edulis (purple type), Passiflora edulis f. flavicarpa (yellow type), Passiflora quadrangularis (giant granadilla), and Passiflora ligularis (sweet granadilla) [15]. In 2023, passion fruit cultivation in India spanned 0.011 million hectares, producing 0.056 million metric tons, predominantly of the yellow and purple types [16]. Beyond its agricultural value, passion fruit holds significant industrial and medicinal importance. The juice [3], leaves (used traditionally in Nagaland for dysentery and hypertension) [17], rind residue (5–6% protein), and seeds (23% oil, comparable to soybean and sunflower oil) [1] are utilized in various applications [18]. In South America, passion fruit is employed as a sedative, diuretic, and treatment for conditions like hypertension, menopause symptoms, and infant colic [19]. Despite its nutritional, industrial, and medicinal potential, passion fruit remains underutilized in India, particularly in the Himalayan region, where the systematic evaluation and physicochemical characterization of diverse genotypes are lacking. Recent studies, such as Patel et al.’s [20] analysis of six genotypes across Passiflora edulis, P. edulis f. flavicarpa, and P. alata in Meghalaya, have begun to address these gaps. The 21st century has seen increased research, technological advancements, and conservation efforts for underutilized crops like passion fruit, highlighting their potential as a diversified income source for farmers due to their short maturity period and high value [21]. This study evaluates fifteen genotypes of Passiflora edulis f. flavicarpa, Passiflora edulis, Passiflora ligularis, and Passiflora quadrangularis collected from Arunachal Pradesh, Manipur, Assam, Tripura, Mizoram, Nagaland, and Sikkim. The morpho-physicochemical properties of their fruit juice, leaves, and tendrils were analysed, and seed protein profiles were validated using SDS-PAGE, contributing to a deeper understanding of their diversity and potential for cultivation and utilization

Passion fruit in Northeastern India exhibits remarkable ecological adaptability, thriving in diverse agro-climatic conditions from tropical lowlands to subtropical highlands. Its resilience to local biotic and abiotic stresses, coupled with high nutritional and industrial value, positions it as a promising crop for sustainable agriculture. An enhanced cultivation of these species could boost local economies by providing income diversification for tribal farmers and supporting agro-based industries. This underscores the need for systematic evaluation to harness their economic potential. Moreover, this study aims to elucidate the genetic variation present within Passiflora species found in the northeastern region of India. The outcomes of this research endeavour will serve as valuable resources for future investigations focused on understanding and harnessing the potential of these species.

2. Materials and Methods

2.1. Details of Genotypes

Fifteen genotypes of Passiflora species were collected from seven states in the eastern region of India, namely Arunachal Pradesh, Assam, Manipur, Nagaland, Mizoram, Sikkim, and Tripura. These genotypes include P. edulis f. flavicarpa Deg, Passiflora edulis Sims, Passiflora ligularis Juss, and Passiflora quadrangularis L. (

Table 1. List of Passiflora edulis f. flavicarpa Deg, Passiflora edulis Sims, Passiflora ligularis Juss, and Passiflora quadrangularis L. and their sources.

Species	Code	Sources	Latitude (N)	Longitude (E)	Altitude
P. edulis f. flavicarpa Deg	P1	Andro, Manipur	24°73′	94°04′	815 m
P. edulis f. flavicarpa Deg	P2	West Imphal, Manipur	24°47′	93°58′	906 m
P. edulis f. flavicarpa Deg	P3	Sutamura, west Tripura, Tripura	23°62′	91°26′	20 m
P. edulis f. flavicarpa Deg	P4	College of Agriculture, Biswanath Cherali, Assam	26°43′	93°08′	82 m
P. edulis f. flavicarpa Deg	P5	Notun Basti, Dimapur, Nagaland	25°55′	93°43′	154 m
P. edulis f. flavicarpa Deg	P6	CHF, Pasighat, Arunachal Pradesh	28°04′	95°19′	162 m
P. edulis Sims	P7	Kangpokpi, Manipur	24°42′	93°46′	1510 m
P. edulis Sims	P8	ICAR-NOFRI, East Sikkim	27°17′	88°36′	882 m
P. edulis Sims	P9	Aizawl, Mizoram	23°43′	92°44′	786 m
P. edulis Sims	P10	CHF, Campus, Pasighat, Arunachal Pradesh	28°04′	95°19′	168 m
P. edulis Sims	P11	Ziro, Lower Subansiri, Arunachal Pradesh	27°32′	93°48′	1566 m
P. edulis Sims	P12	Pasighat, Arunachal Pradesh	28°03′	95°20′	154 m
P. ligularis Juss	P13	Lunghar Village, Ukhrul, Manipur	25°16′	94°42′	1633 m
P. ligularis Juss	P14	Sakhabama, Kohima, Nagaland	25°39′	94°11′	1077 m
P. quadrangularis L.	P15	Pasighat, Arunachal Pradesh	28°03′	95°20′	156 m

, Figure 1).

2.2. Morphological Characterisation

The morphological parameters of leaves, tendrils, and ripe fruits were collected for the study. These consisted of the angle between lateral veins (°), leaf length (cm), leaf width (cm), leaf shape, leaf size, leaf base size, leaf colour, leaf margin shape, the division of leaf lamina, the presence of leaf nectaries, sinus depth, the presence of heterophylly, the presence of stipule, petiole length (cm), tendril length (cm), the length of the right lateral lobe (cm), peduncle length (cm), flower length (cm), filament length (cm), stamen length (cm), the number of flowers per node, fruit length (cm), fruit breadth (cm), fruit weight (g), the number of fruits per vine, fruit yield (kg per vine), peel weight (g), shelf-life (days), the weight of 100 seeds (g), seed length (cm), seed width (cm), the number of seeds per fruit, seed weight per fruit, stem colour, flower colour, petal colour, ovary colour, fruit colour, fruit shape, and seed colour, which were recorded following the morpho-agronomic descriptors for Passiflora spp. [22] (Figure S1 Supplementary Materials) and the tropical fruit descriptor developed by Bioversity International (IPGRI) (Supplementary Materials). The Image J software was used to calculate the angle between lateral veins [23].

2.3. Sample Preparation

The fruits were harvested at full ripeness, retaining their natural skin colour. The fruits were washed in running water, halved, and divided into pulp, peel (albedo and flavedo), and seeds. The parts were packed into plastic bags and stored in a freezer at −20 °C until further analysis.

2.4. Biochemical Characterisation

The biochemical attributes of pulp, leaves, and tendrils were analysed following standard protocol. The total soluble solid (TSS) content in pulp was determined with a °Brix refractometer; total carotenoid content (mg 100 g⁻¹) as per [24]; total flavonoids (mg 100 g⁻¹) as per [25]; antioxidant activity using (2,2-diphenyl-1-picrylhydrazyl, DPPH) (%) [26]; titratable acidity (%) as per [27]; total carbohydrates (%) as per [28]; reducing sugar (%) as per [29], and non-reducing sugar (%) as per [30]. The anthocyanin content (mg 100 g⁻¹) in petioles and tendrils was determined using [31]. The vitamin C (mg 100 g⁻¹) [31], phenol (mg g⁻¹) [25], and chlorophyll content (mg g⁻¹) [32] content in leaves were determined following standard methods (

Table 2. Quality parameters studied following standard protocol.

Plant Part	Characters	References
Leaf	I. Anthocyanin content (mg/100 g)	[31]
	II. Vitamin C content (mg/100 g)	[27]
	III. Phenol content (mg/100 g)	[25]
	IV. Chlorophyll content (mg/g)	[32]
Petiole	V. Anthocyanin content (mg/100 g)	[31]
Tendril	VI. Anthocyanin content (mg/100 g)
Fruit	VII. Vitamin C (mg/100 g)	[27]
	VIII. Total soluble solids (°Brix)	Hand refractometer
	IX. Total carotenoid (mg/100 g)	[24]
	X. Total flavonoids (mg/100 g)	[25]
	XI. Antioxidant activity (DPPH) (%)	[26]
	XII. Titratable acidity (%)	[27]
	XIII. Total carbohydrates (%)	[28]
	XIV. Reducing sugar (%)	[29]
	XV. Non-reducing sugar (%)	[30]

). The absorbance of the solution was measured using Lambda 365, UV—visible spectrophotometer (Perkin Elmer, Waltham, MA, USA).

2.5. Protein Extraction and Estimation

Seeds weighing 0.2 g were extracted from the fully ripened fruits of the selected genotypes of the Passiflora species. These extracted seeds had been pre-soaked in a phosphate buffer solution (pH 7.0) for 24 h before use. Seeds were crushed with a solution of Tris-HCl 0.06 M (PH 7.4), 10 mM urea, 1 mM EDTA, 0.1% TCA, 2.5% glycerol, 0.5% SDS, and 1.25% β-mercaptoethanol. After crushing, the volume was made up to 2 mL and centrifuged at 8000 rpm for 20 min at 4 °C. The estimation of protein was conducted as per Lowry’s method [33] to quantify the amount of protein present in the sample.

2.6. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Electrophoresis was carried out in the presence of denaturing agent (SDS/SLS) as per the procedure described by [34] with some modifications, and RF value and the molecular weight of each protein band were determined to observe variation among the Passiflora species found in the northeastern Himalayan region of India (

Table 3. The formulation for 15% acrylamide separating gel, 5% acrylamide stacking gel, and sample preparation.

	Formulation for 15% Acrylamide Separating Gel	Formulation for 5% Acrylamide Stacking Gel
Water	6.9 mL	5.5 mL
30% Acrylamide mixture	15 mL	1.3 mL
Separating gel buffer (1.5 M Tris-HCl, pH 8.8)	7.5 mL	1.0 mL
2% SDS	0.3 mL	0.1 mL
10% Ammonium persulphate	0.3 mL	0.1 mL
TEMED	0.012 mL	0.008 mL
Sample Preparation
0.6 M Tris-HCl	5.0 mL
1% SDS	0.5 g
0.5% Bromophenol blue solution	5 mL
10% sucrose	5.0 g

).

2.7. Staining Solution

Silver staining was used to stain the protein bands as per [35]; this was performed using fixing solution (50% ethanol, 12% glacial acetic acid, and 0.05 mL formaldehyde), sensitising solution (0.02% aqueous solution of sodium thiosulphate), silver stain solution (0.2% silver nitrate and 0.076% formaldehyde), developing solution (6% sodium carbonate, 0.004% sodium thiosulphate, and 0.05% formaldehyde solution), and terminating/stopping solution (12% acetic acid).

2.8. Statistical Analysis

Data analysis was performed using variance analysis with the SAS software (p < 0.05) version 9.1. In addition, the Pearson correlation matrix and principal component analysis (PCA) were conducted using Python packages, including scikit-learn, bioinfokit, seaborn, scipy, numpy, pandas, and matplotlib. Letters that are the same within a column indicate no significant difference among genotypes. The gels were scored as presence (+) or absence (−) of protein polypeptide bands for protein banding pattern. Depending upon the presence or absence of polypeptide bands, the similarity index (SI) [35] between the genotypes was calculated by the following formula:

$$\mathrm{SI}={\displaystyle \frac{2\mathrm{Z}}{\mathrm{X}+\mathrm{Y}}}\times 100$$

(1)

where Z = the number of similar bands between the genotypes, and X + Y = the total number of bands in the two genotypes compared.

3. Results

3.1. Morphological Variations

Variations in morphological characteristics were evident among the collected genotypes, as illustrated in Figure 1 and detailed in

Table 4. (A,B) Morphological characteristics of fifteen genotypes of Passiflora edulis f. flavicarpa Deg., Passiflora edulis Sims, Passiflora ligularis A. Juss, and Passiflora quadrangularis L.

A
Species	Code	Angle Between Lateral Veins (°)	Leaf Length (cm)	Leaf Width (cm)	Petiole Length (cm)	Tendril Length (cm)	Length of Right Lateral Lobe (cm)	Peduncle Length (cm)	Flower Length (cm)	Filament Length (cm)	Stamen Length (cm)	Number of Flowers per Node
P. edulis f. flavicarpa Deg.	P1	63.38 ab	12.56 bc	16.07 a	2.21 bc	13.39 g	7.17 a	3.27 de	6.93 d	0.83 d	1.80 abc	1.00 b
P. edulis f. flavicarpa Deg.	P2	63.02 ab	12.21 bcd	15.56 ab	1.94 cd	12.70 g	7.03 ab	3.20 de	6.87 d	0.80 d	1.77 abcd	1.00 b
P. edulis f. flavicarpa Deg.	P3	59.70 abc	11.23 cdef	14.10 bcd	1.78 de	16.13 e	6.90 b	3.27 de	5.60 i	0.83 d	1.63 abcde	1.00 b
P. edulis f. flavicarpa Deg.	P4	63.38 ab	12.70 b	15.67 ab	2.22 bc	20.23 c	5.90 de	3.10 de	5.60 i	1.37 a	2.02 a	1.00 b
P. edulis f. flavicarpa Deg.	P5	57.21 bc	10.20 f	12.68 de	1.76 de	14.57 f	6.60 c	3.07 de	6.73 d	0.83 d	1.50 bcdef	1.00 b
P. edulis f. flavicarpa Deg.	P6	42.24 d	11.27 cdef	14.80 abc	3.02 a	22.40 b	5.80 e	3.23 de	7.40 c	0.90 cd	1.67 abcde	1.00 b
P. edulis Sims	P7	58.32 abc	10.68 ef	13.24 cde	1.73 de	20.54 c	5.43 g	2.93 de	6.30 e	1.13 b	0.70 h	1.00 b
P. edulis Sims	P8	57.12 bc	10.86 def	13.92 bcd	1.79 de	19.13 d	5.27 h	3.30 d	5.93 fgh	1.13 b	0.80 gh	1.00 b
P. edulis Sims	P9	57.10 bc	10.66 def	13.80 bcd	1.75 de	19.11 d	5.19 h	3.27 d	5.81 fgh	1.11 b	0.78 gh	1.00 b
P. edulis Sims	P10	57.97 abc	15.13 a	12.07 ef	2.49 b	22.13 b	6.00 d	5.07 c	7.73 b	0.80 d	1.12 fgh	1.00 b
P. edulis Sims	P11	54.07 c	10.41 f	12.64 de	1.96 cd	20.00 c	5.40 gh	2.93 de	5.77 hig	0.67 e	1.28 def	1.00 b
P. edulis Sims	P12	58.79 abc	10.42 f	13.28 cde	1.89 cde	19.20 d	5.30 gh	2.87 e	5.87 fgh	1.13 b	1.39 cdef	1.00 b
P. ligularis Juss	P13	62.17 ab	14.45 a	10.51 fg	2.42 b	14.27 f	0.00 i	7.23 a	6.00 f	0.97 c	1.57 abcdef	1.67 c
P. ligularis Juss	P14	63.55 ab	14.38 a	11.58 ef	1.88 cde	16.13 e	0.00 i	6.43 b	5.97 fg	0.83 d	1.97 ab	1.67 c
P. quadrangularis L.	P15	64.52 a	11.95 bcde	9.44 g	1.56 e	28.13 a	0.00 i	2.40 f	9.20 a	0.83 d	1.42 cdef	2.67 a
B
Species	Code	Fruit Length (cm)	Fruit Breadth (cm)	Fruit Weight (g)	Number of Fruits per Vine	Fruit Yield (kg per vine)	Peel Weight (g)	Shelf-life (days)	Weight of 100 Seeds (g)	Seed Length (cm)	Seed Width (cm)	Number of Seeds per Fruit	Seed Weight per Fruit
P. edulis f. flavicarpa Deg.	P1	6.16 cd	5.33 cd	78.75 b	126.34 ef	9.94 bc	42.85 b	9.33 cde	1.17 f	0.54 cd	0.35 b	146.33 bcd	1.92 ef
P. edulis f. flavicarpa Deg.	P2	6.63 bc	6.16 b	65.1 b	138.33 de	9.01 bcde	42.32 b	8.33 de	0.90 g	0.56 bc	0.38 b	231.33 a	2.08 def
P. edulis f. flavicarpa Deg.	P3	6.16 cd	5.33 cd	69.98 b	144.00 cde	10.07 bc	43.34 b	6.33 e	1.32 f	0.54 cd	0.35 b	193.00 ab	2.54 cdef
P. edulis f. flavicarpa Deg.	P4	6.51 bcd	5.64 bc	77.69 b	118.00 f	9.15 bcd	43.29 b	7.00 e	2.00 de	0.52 de	0.37 b	166.33 bcd	3.03 cd
P. edulis f. flavicarpa Deg.	P5	5.95 cd	5.02 cd	45.18 b	128.00 ef	5.78 cde	20.59 b	11.67 bcd	1.95 de	0.52 de	0.18 c	171.00 bcd	3.27 c
P. edulis f. flavicarpa Deg.	P6	6.51 bcd	5.64 bc	77.69 b	134.92 ef	10.473 b	47.96 b	6.00 e	2.00 de	0.52 de	0.37 b	152.00 bcd	3.04 cd
P. edulis Sims	P7	5.95 cd	5.02 cd	45.18 b	166.67 ab	7.56 bcde	24.59 b	10.33 bcde	2.31 c	0.51 de	0.20 c	140.00 bcd	3.23 c
P. edulis Sims	P8	4.79 e	4.27 ef	32.7 b	159.67 abc	5.22 de	16.30 b	10.00 bcde	1.88 e	0.52 de	0.21 c	123.33 de	2.31 cdef
P. edulis Sims	P9	4.68 e	4.21 f	31.78 b	152.67 bcd	4.85 de	15.90 b	12.00 bcd	1.85 e	0.49 e	0.19 c	87.67 e	1.61 f
P. edulis Sims	P10	6.16 cd	5.33 cd	33.08 b	161.33 abc	5.35 de	16.17 b	9.33 cde	2.17 cd	0.54 cd	0.35 b	146.33 bcd	3.15 cd
P. edulis Sims	P11	5.69 d	4.86 de	43.89 b	157.67 abc	6.95 bcde	22.86 b	9.83 bcde	2.16 cd	0.52 de	0.22 c	136.33 cde	2.97 cde
P. edulis Sims	P12	5.72 d	4.89 de	43.99 b	174.67 a	7.68 bcde	22.23 b	13.00 bc	2.18 cd	0.51 de	0.20 c	141.67 bcd	2.92 cde
P. ligularis Juss	P13	7.17 b	5.38 cd	53.41 b	86.67 g	4.62 e	32.31 b	14.00 b	3.20 b	0.60 b	0.17 c	164.33 bcd	5.27 b
P. ligularis Juss	P14	7.16 b	5.36 cd	53.23 b	87.33 g	4.67 e	31.94 b	12.00 bcd	3.20 b	0.60 b	0.17 c	180.33 bc	5.25 b
P. quadrangularis L.	P15	14.48 a	9.30 a	496.67 a	52.33 h	26.23 a	360.00 a	27.33 a	5.45 a	0.79 a	0.62 a	172.33 bcd	9.37 a

The same letter in the column indicates no significant difference.

(Parts A and B). The genotype P10 of P. edulis Sims displayed the highest leaf length at 15.13 cm, while genotypes P13 (14.45 cm) and P14 (14.38 cm), belonging to P. ligularis Juss., exhibited similar leaf lengths. Notably, the maximum leaf width was recorded in genotype P1 (16.07 cm), followed by P4 (15.67 cm) and P2 (15.56 cm), all of which are classified under Passiflora edulis f. flavicarpa Deg. In terms of flower length, genotype P15 of Passiflora quadrangularis L. displayed the longest at 9.20 cm, followed by P10 (7.73 cm) of Passiflora edulis Sims, which was comparable to P10 (7.40 cm) of Passiflora edulis f. flavicarpa Deg. The genotype P4 of Passiflora edulis f. flavicarpa Deg. exhibited the longest stamen and filament lengths, measuring 2.02 cm and 1.37 cm, respectively. Conversely, genotype P15 of Passiflora quadrangularis L. demonstrated superior metrics in various parameters, including fruit length (14.48 cm), breadth (9.30 cm), fruit weight (496.67 g), seed length (0.79 cm), the angle between lateral veins (64.52°), seed width (0.62 cm), seed weight per fruit (9.37 g), and shelf life of fruit (27 days at room temperature), when compared to Passiflora edulis f. flavicarpa Deg., Passiflora edulis Sims, and Passiflora ligularis Juss genotypes under study. Furthermore, fruit shapes exhibited diversity ranging from oblate (genotypes P1, P2, P3, P4, P6, P7, P8, P9, P10, P11, and P12 of Passiflora edulis f. flavicarpa Deg.) to ellipsoid (genotype P5 of Passiflora edulis f. flavicarpa Deg., and genotypes P13 and P14 of Passiflora ligularis Juss), and oblong (genotype P15 of Passiflora quadrangularis L.). The length of the right lateral lobe varied significantly, with Passiflora edulis f. flavicarpa Deg. (genotype P1) exhibiting the maximum at 7.17 cm, while being absent in genotypes P13 and P14 of Passiflora ligularis Juss and genotype P15 of Passiflora quadrangularis L. Notably, a higher number of fruits per vine was observed for genotypes P12 (174.67) and P7 (166.67), both belonging to Passiflora edulis f. flavicarpa Deg. In contrast, genotype P15 (52.33) of Passiflora quadrangularis L. exhibited the least. Genotype P15 displayed the maximum number of flowers per node (2.67), followed by genotypes P13 and P14 (1.67), while other genotypes bore only one flower per node. In terms of fruit yield (kg per vine), genotype P15 (26.23 kg per vine) of Passiflora quadrangularis L. and genotype P6 (10.47 kg per vine) of Passiflora edulis f. flavicarpa Deg. demonstrated the highest yields. In contrast, genotype P13 (4.62 kg per vine) of Passiflora ligularis Juss exhibited the lowest.

3.2. Contributions of Morphological Characteristics Towards Diversity in Passiflora Species

The distinct morphological parameters such as angle between lateral veins (°), leaf length (cm), leaf width (cm), petiole length (cm), tendril length (cm), the length of the right lateral lobe (cm), peduncle length (cm), flower length (cm), filament length (cm), stamen length (cm), the number of flowers per node, fruit length (cm), fruit breadth (cm), fruit weight (g), the number of fruits per vine, fruit yield (kg per vine), peel weight (g), the weight of 100 seeds (g), seed length (cm), seed width (cm), the number of seeds per fruit, and seed weight per fruit revealed diversity in Passiflora species. Among the different variables, seed length contributed the highest percentage towards genetic divergence (55.24%), followed by the seed weight per fruit (22.86%), number of seeds per fruit (8.57%), and filament length (1.90%). In contrast, the length of the right lateral lobe and seed breadth (5.71%) contributed to the same percentage (Figure 2).

3.3. Biochemical Characteristics of Fruit Juice, Leaves, Petioles, and Tendrils

Significant differences were observed in the biochemical attributes of fruit juice, leaves, tendrils, and petioles among Passiflora edulis f. flavicarpa Deg., Passiflora edulis Sims, Passiflora ligularis A. Juss and Passiflora quadrangularis L. collected from the northeastern Himalayan region of India under study (

Table 5. Biochemical parameters of fruit juice of Passiflora edulis f. flavicarpa Deg., Passiflora edulis Sims, Passiflora ligularis A. Juss, and Passiflora quadrangularis L.

Species	Genotypes	Vit C (mg g−1)	Total Soluble Solids (°Brix)	Total Carotenoids (mg g−1)	Total Flavonoids (mg g−1)	Antioxidant Activity (DPPH) (%)	Titratable Acidity (%)	Total Carbohydrate (%)	Reducing Sugar (%)	Non-Reducing Sugar (%)	Fruit Juice Content (mL/fruit)
P. edulis f. flavicarpa Deg.	P1	0.238 bcd	16.17 d	0.100 hi	0.114 def	10.85 cde	3.56 ab	10.14 de	4.92 cdef	5.20 abcd	34.17 b
P. edulis f. flavicarpa Deg.	P2	0.26.1 abc	15.97 d	0.090 ij	0.113 def	11.70 bcd	3.91 a	11.14 abcd	4.63 defg	6.54 a	20.70 bcd
P. edulis f. flavicarpa Deg.	P3	0.262 abc	15.13 e	0.116 h	0.09 ef	12.93 bcd	3.41 abc	12.12 ab	4.93 cdef	5.79 abc	24.09 bcd
P. edulis f. flavicarpa Deg.	P4	0.262 abc	16.03 d	0.08.7 ij	0.11 def	11.48 bcd	3.32 bc	10.31 cde	4.10 fg	6.52 a	31.35 bc
P. edulis f. flavicarpa Deg.	P5	0.265 abc	18.13 a	0.300 c	0.35 a	22.15 a	1.15 gh	10.84 bcd	6.88 a	4.72 abcd	21.31 bcd
P. edulis f. flavicarpa Deg.	P6	0.214 cd	17.53 c	0.077 j	0.16 cde	12.50 bcd	2.43 e	7.81 f	4.35 efg	3.05 d	34.20 b
P. edulis Sims	P7	0.292 ab	18.07 a	0.265 f	0.11 def	11.96 bcd	2.78 de	12.36 ab	6.36 ab	6.28 ab	17.35 d
P. edulis Sims	P8	0.226 cd	18.28 a	0.397 a	0.24 bc	12.60 bcd	2.59 e	10.59 bcd	6.92 a	3.42 cd	14.09 d
P. edulis Sims	P9	0.207 cd	17.94 ab	0.196 g	0.07 g	9.80 def	2.52 e	12.06 abc	5.54 bcd	5.99 abc	10.94 d
P. edulis Sims	P10	0.214 cd	17.27 c	0.240 b	0.26 b	14.76 b	3.19 bcd	12.88 a	5.98 abc	6.40 ab	13.75 d
P. edulis Sims	P11	0.320 a	14.70 ef	0.285 e	0.10 ef	13.70 bc	2.91 cde	12.18 ab	6.41 ab	5.96 abc	18.14 cd
P. edulis Sims	P12	0.177 de	17.53 bc	0.145 d	0.20 bcd	10.33 cdef	1.20 g	10.14 de	5.35 bcde	3.86 bcd	18.78 cd
P. ligularis Juss	P13	0.134 e	14.43 f	0.0001 k	0.11 ef	7.52 efg	0.64 h	8.41 f	3.51 g	4.25 abcd	16.77 d
P. ligularis Juss	P14	0.127 e	14.64 bc	0.0001 k	0.11 def	7.17 fg	0.63 h	8.80 ef	4.03 fg	4.50 abcd	16.77 d
P. quadrangularis L.	P15	0.308 a	13.54 g	0.0172 k	0.17 cde	6.28 g	1.81 f	10.32 cde	5.52 bcde	4.92 abcd	117.92 a

The same letter in the column indicates no significant difference.

and

Table 6. Biochemical parameters in leaves, petioles, and tendrils of Passiflora edulis f. flavicarpa Deg., Passiflora edulis Sims, Passiflora ligularis A. Juss, and Passiflora quadrangularis L.

Species	Genotypes	Leaf Vit. C (mg g−1)	Leaf Phenol (mg g−1)	Leaf Chlorophyll (mg g−1)	Petiole Anthocyanin (µg g−1)	Tendril Anthocyanin (µg g−1)
P. edulis f. flavicarpa Deg.	P1	1.151 cd	1.538 h	2.84 ab	20.30 bcde	28.90 ab
P. edulis f. flavicarpa Deg.	P2	1.1807 c	1.527 h	2.90 a	18.60 cde	33.90 a
P. edulis f. flavicarpa Deg.	P3	0.981 de	1.432 h	1.56 de	30.50 ab	26.90 abc
P. edulis f. flavicarpa Deg.	P4	1.218 c	1.795 g	1.50 de	15.70 def	19.80 abcd
P. edulis f. flavicarpa Deg.	P5	1.748 a	1.942 g	1.87 cde	28.10 abc	21.40 abcd
P. edulis f. flavicarpa Deg.	P6	1.256 c	1.798 g	1.16 e	9.30 ef	24.50 abcd
P. edulis Sims	P7	1.172 cd	2.709 e	2.16 bcd	19.0 cde	31.40 ab
P. edulis Sims	P8	1.101 cd	2.813 de	1.57 de	35.40 a	21.10 abcd
P. edulis Sims	P9	1.183 c	2.242 f	1.64 cde	5.90 f	20.40 abcd
P. edulis Sims	P10	489 g	4.985 a	2.60 ab	10.10 def	31.10 ab
P. edulis Sims	P11	1.258 c	2.968 cd	1.32 e	4.30 f	14.10 bcd
P. edulis Sims	P12	1.500 b	3.059 c	2.38 abc	5.90 f	6.70 d
P. ligularis Juss	P13	0.882 ef	3.108 c	1.23 e	21.50 bcd	26.40 abc
P. ligularis Juss	P14	0.752 f	3.113 c	1.10 e	20.40 bcde	16.0 abcd
P. quadrangularis L.	P15	0.493 g	3.489 b	1.65 cde	17.80 cde	9.30 cd

The same letter in the column indicates no significant difference.

). Among the horticultural characteristics, a significant result was recorded for maximum juice content (mL per fruit) in genotype P15 (117.92 mL) followed by P6 (34.20 mL). In comparison, the lowest value was registered in genotype P9 (10.94 mL). The highest TSS content was recorded for P8 (18.28 °Brix), a purple type (Passiflora edulis Sims), and P5 (18.13 °Brix), a yellow type (Passiflora edulis f. flavicarpa Deg.). In contrast, the lowest TSS was recorded for P15 (13.54 °Brix), a giant granadilla (Passiflora quadrangularis L.). The highest titratable acidity content was recorded for P2 (3.91%) and P1 (3.56%), while the lowest was recorded in P14 (0.63%). The highest vitamin C content in fruit juice was recorded in P11 (0.320 mg g⁻¹) and P15 (0.309 mg g⁻¹), whereas the lowest values were recorded for P13 (0.134 mg g⁻¹) and P14 (0.128 g⁻¹). The genotype P10 contained the highest amount of total carbohydrates (12.88%), followed by P7 (12.36%), while the lowest value was recorded for P6 (7.81%). The highest percentage of reducing sugar was recorded in genotype P8 (6.92%), which was comparable to P5 (6.88%), while the lowest was registered in genotype P13 (3.51%) of Passiflora ligularis Juss. The highest percentage of non-reducing sugar was recorded in P2 (6.54%) and P4 (6.52%), while the lowest was in P6 (3.05%) of Passiflora edulis f. flavicarpa Deg.

Among the different genotypes, the total carotenoid content was highest in genotypes P8 (0.397 mg g⁻¹) and P5 (0.30 mg g⁻¹). Both genotypes belong to Passiflora edulis Sims, while the lowest was registered in genotypes P13 and P14 (0.0001 mg g⁻¹), and both species belong to Passiflora ligularis Juss. The genotype P5 contained a significantly higher amount of total flavonoid and antioxidant activity (0.355 mg g⁻¹; 22.15%), followed by P10 (0.261 mg g⁻¹; 14.76%), while the lowest was registered in genotype P9 (0.077 mg g⁻¹) for flavonoids and genotype P15 (6.28%) for antioxidant activity. The maximum vitamin C content of leaves was recorded for P5 (1.749 mg g⁻¹) and P12 (1.500 mg g⁻¹), while it was the least in genotype P10 (0.489 mg g⁻¹). Significantly higher phenol content in the leaves was recorded for P10 (4.985 mg g⁻¹), P15 (3.489 mg g⁻¹), P14 (3.113 mg g⁻¹), and P13 (3.108 mg g⁻¹) while the least was found in genotype P3 (1.432 mg g⁻¹). The maximum total chlorophyll content of leaves was recorded for P2 (2.91 mgg⁻¹),

P1 (2.84 mg g⁻¹), and P10 (2.60 mg g⁻¹) while the least was found in P13 (0.0012 mg g⁻¹). The anthocyanin in petioles of P8 (35.40 µg g⁻¹), P3 (30.50 µg g⁻¹), and P5 (28.10 µg g⁻¹) contained a significantly higher amount. In comparison, genotype P2 showed a higher amount of anthocyanin in tendrils (33.90 µg g⁻¹), followed by P7 (31.40 µg g⁻¹) and P10 (31.1 µg g⁻¹). The lowest anthocyanin content was recorded in genotype P11 (4.30 µg g⁻¹) in petioles and genotype P12 (6.70 µg g⁻¹) in tendrils.

3.4. Total Flavonoids Share the Highest Percentage Towards Diversity in Passion Fruit Species

The biochemical parameters such as vitamin C, total soluble solids, total carotenoid, total flavonoids, antioxidant activity, titratable acidity, total carbohydrates, reducing sugar, non-reducing sugar in fruit juice; anthocyanin content in leaves, petioles, and tendrils; and phenol, total chlorophyll, and vitamin C content in leaves had diverse contributions. Among the traits studied, total flavonoid content contributed the highest percentage towards genetic divergence (63.81%), followed by chlorophyll content in leaves (17.14%), total carbohydrates content (8.57%), anthocyanin content in petioles (3.81%), and total carotenoid content (1.90%) (Figure 3).

3.5. Characterisation of Passiflora Species Through Seed Protein Profiles (SDS-PAGE)

The protein banding pattern of fifteen genotypes was generated by SDS-PAGE (Figure 4). A cluster analysis of the banding pattern based on similarity index and UPGMA resulted in distinct clusters (Figure 4b). A total of 89 protein bands, as per

Rm values, were identified through silver staining. The genotypes displayed significant variation in the number of protein bands, ranging from six to eleven. Among them, P13 and P14 showed the highest number (11) of protein bands, while the lowest number (6) of bands was present in P1 (P. edulis f. flavicarpa Deg.). P. ligularis Juss (genotypes P13 and P14) was characterised by three specific protein bands at bands 2, 10, and 13. In contrast, band number 30 was found only in P1 (P. edulis f. Flavicarpa Deg.) and P13 (P. ligularis Juss). Band number 2 (Rm = 0.11) was exclusively present in genotype P8; similarly, band number 9 (Rm = 0.18) was found in genotypes P11 and P14, whereas it was absent in all other genotypes studied. The same protein bands were observed in P. edulis f. Flavicarpa Deg. (P2, P3, and P4) and P. edulis Sims (P8, P9, P10, and P11).

3.6. Correlation Analysis of Different Traits

Among the morphological traits studied, the fruit length was maximally positively correlated with fruit breadth, fruit weight, peel weight, and seed length; fruit weight with fruit yield and peel weight; and the weight of 100 seeds with seed weight/fruit. On the other hand, the number of fruits/vine was maximally negatively correlated with seed weight/fruit; fruit length, breadth, and weight with number of fruits/vine; the weight of 100 seeds, seed length, seed weight/fruit, and angle between lateral veins with leaf width and the length of the right lateral lobe; flower length with filament length; the length of the right lateral lobe with the number of inflorescence/node and peduncle length; tendril length with stamen length, peduncle length, and the length of the right lateral lobe, seed weight with leaf width and the length of the right lateral lobe; and fruit yield with pedicle length, the length of the right lateral lobe, leaf width, and petiole length. The number of fruits/vine had a negative correlation with most of the studied parameters except for the length of the right lateral lobe, leaf width, and filament length (Figure 5). Regarding biochemical traits, the total carotenoid content was significantly positively correlated with reducing sugar, total flavonoids, and antioxidant activity; and total soluble sugar with the total carotenoids, total flavonoids, and antioxidant activity of fruit juice as well leaf vitamin C content. However, leaf vitamin C content showed a negative correlation with leaf phenol content, while total soluble sugar content showed a maximum negative correlation with fruit juice content (Figure 5B).

3.7. Principal Components Analysis of Different Traits

The principal component analysis was performed for morphological and biochemical traits across the genotypes to understand the relationship between critical variables (Figure 6A,B). Principal component 1 accounted for 47.7% of the variation, while PC2 accounted for 13.8% of the variation in the morphological traits studied. Petiole length exhibited a negative correlation with P. edulis f. flavicarpa Deg. (P1) and P. edulis Sims (P7 and P10) in PC1, whereas most traits showed a positive correlation in PC2 in other passion fruit genotypes under study. Regarding biochemical traits, a total variation of 43% was accounted for, with most traits showing a positive correlation. The genotype P5 (P. edulis f. flavicarpa Deg.) accounted for total soluble sugar while genotypes P1 (P. edulis f. flavicarpa Deg.), P14 (P. ligularis Juss), and P15 (P. quadrangularis L.) were associated with fruit juice content.

4. Discussion

Understanding the genetic diversity within collections of genotypes is crucial for effectively conserving and managing germplasm resources and utilising them in breeding programs for passion fruit [36]. Genetic variation among genotypes is essential for developing new crop varieties that enhance productivity and resilience against biotic and abiotic stresses. In this study, different morpho-physicochemical traits and seed proteins were recorded to estimate diversity among fifteen genotypes belonging to P. edulis f. flavicarpa Deg., P. edulis Sims, P. ligularis Juss, and P. quadrangularis L., collected from Arunachal Pradesh, Manipur, Assam, Tripura, Mizoram, Nagaland, Sikkim, and Mizoram states located in the northeastern Himalayan region of India during 2018–2020.

4.1. Morphological Characterisation

Significant morphological variation was observed among Passiflora genotypes, attributable to their diverse species origins and genetic backgrounds. Notably, within Passiflora edulis Sims. (genotype P10), intraspecific variations were evident, likely due to the heterogeneous seed sources. This aligns with previous studies by [17,37], which reported considerable morphological diversity among Passiflora species. Among the genotypes, Passiflora quadrangularis L. (genotype P15) exhibited superior traits, including maximum fruit length, breadth, weight, seed length, seed width, seed weight per fruit, and angle between lateral veins, compared to Passiflora edulis f. flavicarpa Deg., Passiflora edulis Sims., and Passiflora ligularis Juss. These characteristics reflect the inherently larger size of P. quadrangularis L., as corroborated by [38]. In terms of floral morphology, P. quadrangularis L. displayed the longest flowers, while P. edulis f. flavicarpa showed the greatest stamen and filament lengths, consistent with findings by [39]. Such morphological diversity has practical implications for breeding and market preferences. For instance, specific Passiflora species may be favoured for particular culinary or commercial applications due to their distinct fruit sizes and shapes. Additionally, environmental factors, cultural practices, and genotype selection significantly influence fruit size and yield [40]. These findings underscore the importance of selecting elite genotypes with enhanced fruit yield and quality to meet the demands of growers and breeders aiming to optimize Passiflora cultivation.

4.2. Biochemical Characterisation

The biochemical profiles of Passiflora species from the northeastern Himalayan region revealed substantial diversity in nutritional and bioactive compounds, highlighting their potential for functional food development and the conservation of underutilized species. The highest total soluble solid (TSS) content was recorded in genotype P8 (P. edulis Sims., purple type, 18.28 °Brix) and P5 (P. edulis f. flavicarpa Deg., yellow type, 18.13 °Brix), while the lowest was observed in P15 (P. quadrangularis L., 13.54 °Brix). These results align with [20,41], who noted that TSS content varies with species, geographical origin, genetic factors, environmental conditions, and harvesting practices. For example, genotype P8, collected from East Sikkim at 882 m altitude, exhibited elevated TSS, likely due to favourable microclimatic conditions. High TSS content enhances flavour, marketability, and nutritional value, making genotypes like P8 and P5 highly desirable for the passion fruit industry [42]. Ascorbic acid (vitamin C) content further underscores the nutritional potential of Passiflora species. Both fruit juice and leaves were rich in vitamin C, with genotype P5 (P. edulis f. flavicarpa) recording the highest leaf ascorbic acid content. This supports the use of Passiflora leaves as a valuable source of vitamin C for nutritional, medicinal, and commercial applications, as noted by [43]. Traditionally, Passiflora leaves have been used to treat ailments such as dysentery and hypertension, and they exhibit sedative, diuretic, anti-helminthic, anti-diarrheal, and stimulant properties [18,19]. The antioxidant potential of these genotypes, driven by high vitamin C and total carotenoid content, was particularly pronounced in P8 and P5, consistent with [12].

Flavonoid content, known for its antioxidant, pharmacological, and cytotoxic properties [44], was highest in genotypes P5 (P. edulis f. flavicarpa) and P10 (P. edulis Sims.). These values (approximately 0.29 mg catechin g⁻¹ for P5 and 0.28 mg catechin g⁻¹ for P10) are comparable to those reported by [45] for similar species. The elevated flavonoid levels in P5 and P10 suggest their suitability for breeding programs targeting enhanced bioactive profiles. Antioxidant activity, measured via DPPH assay, was highest in P5, corroborating the correlation between carotenoid content and antioxidant capacity reported by [12]. This synergy of bioactive compounds, including phenolics, carotenoids, and flavonoids, contributes to the overall antioxidant potential, as highlighted by [46]. These findings are consistent with [47,48], who documented significant antioxidant activity in P. edulis leaves.

The giant granadilla (P. quadrangularis L.) offers additional nutritional versatility, as its green fruits can be used as a fresh vegetable [49]. Meanwhile, the leaves of P. edulis f. flavicarpa and P. edulis Sims. serve as a rich vitamin C source, enhancing their potential in both dietary and therapeutic applications. The observed biochemical variability is influenced by factors such as fruit maturation stage, crop year, variety, storage conditions, and temperature, as reported by [50,51]. For instance, total sugar content in yellow passion fruit ranged from 5.75 to 13.97 g/100 g across different locations, aligning with our findings and reflecting the impact of geographical variation [52].

4.3. Seed Protein Profiling

Seed protein profiling via SDS-PAGE revealed significant genetic diversity among Passiflora genotypes, supporting their use as genetic markers in breeding programs. Seed proteins, being direct products of structural genes, reflect genetic variations accurately [53,54]. Modifications in gene coding sequences typically manifest as alterations in the primary structure of the encoded proteins [55]. Our analysis identified a low total frequency (TF%) of 40% in P. edulis varieties, suggesting evolutionary divergence among cultivated taxa, as noted by [14]. Unique protein bands at 65.66 kDa in P. foetida and 17.50 kDa in P. edulis confirmed interspecific variation. The greatest genetic distance was observed between genotypes P3 (P. edulis f. flavicarpa) and P14 (P. ligularis), indicating their potential for hybridization in breeding programs to enhance genetic diversity.

4.4. Correlation and PCA

The correlation analysis of morphological and biochemical traits in passion fruit reveals key relationships guiding breeding strategies. Fruit length showed strong positive correlations with fruit breadth, weight, peel weight, and seed length, indicating their role in fruit size and quality [56]. Fruit weight positively correlated with yield, suggesting larger fruits enhance productivity [12]. The positive link between 100-seed weight and seed weight per fruit underscores seed traits’ importance in fruit development. Negative correlations between fruit number per vine and seed weight per fruit, fruit length, breadth, and weight suggest a trade-off between fruit quantity and size [17]. Negative associations of tendril length, seed weight, and yield with leaf width, right lateral lobe length, and peduncle length indicate resource competition between vegetative and reproductive traits [57]. The total carotenoid content positively correlated with reducing sugar, total flavonoids, and antioxidant activity, crucial for nutritional quality [52]. However, leaf vitamin C negatively correlated with phenol content, suggesting metabolic trade-offs. These findings support breeding for larger fruits and enhanced biochemical traits while balancing fruit number for optimal yield.

The principal component analysis (PCA) revealed significant insights into the morphological and biochemical diversity among passion fruit genotypes. PC1 and PC2 collectively explained 61.5% of the variation in morphological traits, with PC1 (47.7%) indicating a dominant influence of key traits. The negative correlation of petiole length with P. edulis f. flavicarpa Deg. (P1) and P. edulis Sims (P7 and P10) in PC1 suggests these genotypes may share distinct structural adaptations, potentially linked to environmental or genetic factors affecting petiole development [17]. Conversely, the positive correlation of most morphological traits in PC2 across other genotypes highlights broader morphological diversity, consistent with findings in Passiflora species where traits like leaf and stem morphology vary significantly [58]. For biochemical traits, the PCA accounted for 43% of the variation, with most traits positively correlated, indicating coordinated biochemical expression across genotypes. The high total soluble sugar content in P. edulis f. flavicarpa Deg. (P5) aligns with its known suitability for juice production, as reported by [52]. Similarly, the association of P. edulis f. flavicarpa Deg. (P1), P. ligularis Juss (P14), and P. quadrangularis L. (P15) with fruit juice content underscores their potential for commercial applications, corroborating studies on juice yield in Passiflora [12]. These results suggest that targeted breeding for specific morphological and biochemical traits could enhance passion fruit quality and yield.

5. Conclusions

From this study, it can be concluded that fifteen genotypes from four Passiflora species collected from various parts of the northeastern region of India exhibit significant variation in morphological and biochemical characteristics, comprising thirty-nine morphological traits and seventeen biochemical characters. SDS-PAGE protein profiling also revealed variations at both the intra- and interspecific levels among Passiflora species found in northeast India. Based on the mean performance of all of the traits studied, genotypes P2, P8, P3, P5, P11, and P6 were identified as superior for most of the yield components and fruit quality traits. These genotypes could serve as valuable parental sources in future breeding programs.