Contribution of Root Anatomical Characteristics in Fruit Profile of Pomegranate Genotypes to Expand Production Area in Pakistan

Abstract

Pomegranate adaptation to abiotic stress conditions has led to its wide dispersion in Pakistan and to the appearance of new, local genotypes. These genotypes are important to characterize for breeding programs aimed towards the production of new cultivars suitable to arid, semi-arid, and moderate weather regions. In this study, eleven pomegranate accessions were investigated for fruit morphological and biochemical traits, and root anatomic adaptation under diverse climates. The commercial accession (Sava) had the maximum fruit weight (373.97 g) compared to accessions KK-I (60.94 g) and KK-II (71.63 g), which were old plantations established since United India. Most of the accessions were juicy with a wood portion index (WPI) ranging from 0.93 to 1.06%; however, the non-commercial accession of Khushab Kanhatti (KK-I) showed the highest WPI (4.38%). A high variation occurred among all accessions for total soluble solids, total sugars, and total ascorbic acid contents with a maximum in accession Sava (16.46 °Brix), TK-II (53.16%), and QW-I (0.68%), respectively. Root anatomical studies in accessions depicted significantly high variation, and accession Sava of semi-arid climate produced maximum root epidermis (97.43 µm²), phloem (2730.78 µm²), and metaxylem (717.77 µm²) area. In contrast, high cortex thickness (104.74 µm), cortex area (462.74 µm²), and vascular area (114,382.10 µm²) was measured in accessions KK-II and KK-III of Khushab district with an arid climate. The germplasm of diverse regions showed a strong association and clustered into two main classes based on fruit morpho-chemical and root anatomical characteristics. These traits are likely to provide clues towards plants adaptation to various growing conditions and can be exploited successfully in crop improvement programs.

1. Introduction

The place of origin of pomegranate (Punica granatum L.) is Central Asia, from where it has spread to the rest of the world. Later, it was domesticated and cultivated in Persia, India, and various coastal Mediterranean regions about 4000 years ago and is now commercially grown throughout the Mediterranean region of Asia, Europe, Africa, Afghanistan, China, Spain, and the USA [1,2]. Iran, Afghanistan, India, Spain, Turkey, and Egypt are leading pomegranate producing countries [3], contributing 3086 thousand tons of total world production from an area of 300 thousand hectares [4]. Pomegranate plant parts (leaf, root, and flowers) and fruits have high nutritional content like antioxidants, anthocyanin, vitamins, minerals, tannins, and various polyphenols of vast medicinal applications [5], and thus they are known as super fruits and fruits of love. Significant variations exist in commercially grown and non-commercial domesticated pomegranates (likewise wild) for various fruit quality-related characters, i.e., fruit weight, rind weight and thickness, aril juice contents, acidity, sweetness, and wood portion index [6]. Pomegranate fruits are divided into hard, semi-hard, soft, and semi-soft based on their wood portion index (WPI) [7]. Commercial pomegranate varieties are propagated through stem cuttings [8]; however, root initiation and growth are promoted by treating the cuttings with auxins [8,9].

Plants depend on their roots for moisture, mineral elements, and climate change adaptation, which are necessary for their survival, yield, and nutritional quality. Hence, the study of anatomical features is useful in characterization, identification, and diversity estimation [10,11]. Organizational alteration in root structure is important to estimate the ability of tolerance in cultivars exposed to diverse agro-climatic stresses [12]. Abiotic factors cause structural, volume, and natural changes in root tissues, resulting in survival under unfavorable edaphic and environmental conditions [13]. Parenchyma tissues increase the diffusion of photosynthetic and atmospheric oxygen, variation in the root cortex, and control the water and nutrient transport [12]. The endodermis and exodermis parts of the roots work as an apoplastic barrier for selective transport [14,15]. Taxonomic problems and habitat ecology of certain plant germplasm and grass species are successfully resolved through structural studies of roots [16,17,18] Similarly, diversity in root anatomical studies of 22 date palm cultivars proved the reasoning of its adaptability in diverse ecological regions of the world [11]. Root features are adversely affected by diverse agro-climatic conditions with a reduction in xylem vessel area, which affects plant physiology and biomass [17].

Annual pomegranate production in Pakistan is 57.8 thousand tons on an area of 14.9 thousand hectares [19] and ranks at the 10th position worldwide; however, the productivity is quite low (3.88 tons/ha.). Some important locally marketed pomegranate cultivars are Sandhora, Sava (white arils), Kalahari (Pinkish white arils), Be-danna (soft seeded red and white arils), Kandahari, and Tarnab-Gulabi. Though the crops have a long history of existence in the warm temperate Himalayan range and commercially produced in tropical to subtropical, arid to semi-arid and desert zones, it is still a minor and neglected fruit crop with little research on germplasm characterization and ecological structural changes. Therefore, considering the above discussion, the objective of this study was to unveil the root anatomy of 11 cultivated and non-commercial pomegranate genotypes to assess the adaptation of pomegranate plantation under diverse agro-climatic zones of Pakistan to expand its supply window.

2. Materials and Methods

2.1. Materials

The plants of 11 selected pomegranate genotypes were in situ and grew in 6 selected regions of Pakistan, as detailed in

Table 1. Genotype characters, code and GPS data of eleven pomegranate genotypes collected from six diverse locations.

Accession Code	Collection Districts	Climatic Conditions	Accession Local Name	Market Value	Collection Site	Qualitative Genotype Traits
TG	Peshawar (Tarnab)	Cool temperate	Tarnab Gulabi	Commercial	Agri. Res. Institute, Tarnab	Commercial variety with large size red fruit and sweet juicy red arils
TK-I			Tarnab Kandahari-I			Large size red fruit with less juice and white arils
TK-II			Tarnab Kandahari-II	Non-commercial		Large size red fruit with less juice and white arils
KK-I	Khushab (Kanhati- Garden	Semi-arid	Kanhati-green	Non-commercial	Kanhatti Garden, Khushab	Small size greenish fruit with white pink sour arils
KK-II			Kanhati-Sandhora			Medium size light green fruit with white pink sweet arils
KK-III			Kanhati-Sava	Commercial		Large size yellowish red fruit with pink red sweet arils
CD-I	Chakwal	Semi-arid	Chakwal-green	Non-commercial	D.C.O House, Chakwal	Small size green color fruit with white pink arils, sour in taste with less juice.
CD-II			Chakwal-Lalari			Small size reddish fruit with red sour arils
BP-G	Bahawalpur	Arid	Bahawalpur green	Non-commercial	Regional Agri. Res. Inst., Bahawalpur	Small size fruit, green in color with white arils, sour in taste with less juice
Sava	Muzaffargarh	Sub-tropical	Sava	Commercial	Alipur (Nabi Shah Wala)	Large size light green fruit with sweet and juicy white arils
QW-I	Quetta	Hot temperate	White Kagzi	Commercial	Barkhan Balochistan	Large size red fruit with sweet whitish pink arils

TG: Tarnab Gulabi; TK-I: Tarnab Kandhari-I; TK-II: Tarnab Kandhari-II; KK-I, II & III: Khushab accessions; DC-I & DC-II: accessions growing in DC house Chakwal; BP-G: Bahawalpur green; Sava: commercial genotype of Muzaffargarh; QW-I: Kandhari white.

. Hard wood cuttings of these plants were collected from these regions during the dormant season to record various root anatomical data. Cuttings were shown on well prepared ridges of clay loam soil, enriched with farm yard manure (FYM) at experimental area of Fruit Plant Nursery, Institute of Horticultural Sciences to get root germination, and root anatomical studies were performed at the Taxonomy Laboratory, Department of Botany, University of Agriculture, Faisalabad. Fruit samples of selected genotypes were also collected from these regions to record fruit morphological data to access variation in selected genotypes. Details of the qualitative traits of selected genotypes [20] are described in Table 1, and GPS data of the study area is marked on the map of Pakistan as shown in Figure 1.

2.2. Methods

2.2.1. Fruit Morphological and Biochemical Studies

Fruit samples collected from selected plants of 11 pomegranate genotypes were used to record the weight and diameter of fruits, arils, seeds, and crown; rind weight and thickness were recorded with a Vernier caliper (Model KBD-MT 0014) following the pomegranate plant descriptor. The wood portion index (WPI) indicates the juiciness of arils; high WPI means less juiciness and high proportion of wood in the aril. It was recorded through binary observation of the selected panel through a questionnaire.

Wood portion index (WPI) or juiciness of selected pomegranate genotypes were screened as soft-or hard-seeded following the formula of Martinez et al. [21] as follows:

Wood Portion Index (WPI) = (100 seeds weight in gram/100 aril weight in gram) × 100

(1)

Total soluble solids in the aril juice of each of the 11 selected pomegranate genotypes were measured in a digital refractometer (ATAGO RX 5000, Japan Development Assistance) and titratable acidity in aril juice was measured by following standard titration methods [22].

Water-dissolved aril juice samples were prepared following the method as described by Razzaq et al. [23] to measure total soluble sugars [24] and ascorbic acid contents [25] in a spectrophotometer.

2.2.2. Root Anatomical Studies of Selected Pomegranate Genotypes

Preservation, sectioning, staining, and mounting: Root samples (2 cm) were preserved in a formalin acetic alcohol (FAA) solution containing 5% formalin, 10% acetic acid, 50% ethanol, and 35% distilled water. Thereafter, the preserved material was subsequently transferred to an acetic alcohol solution (acetic acid 25% v/v, ethanol 75%) for the long term preservation of root samples [26].

A free-hand sectioning technique was used to prepare permanent slides of root transverse sections cut with the razor blade, and some fine sections were carefully picked up on wash glass for staining and dehydration through a series of washings with ethanol (30%, 50%, and then 70% for 15 min each). For staining, the lignified tissues (xylem vessels and sclerenchyma) were transferred to safranin (1.0 g dissolved in 100 mL, 70% alcohol) for 20 min, dehydrated in 90% alcohol for 5 min, and stained with fast green (1.0 g dissolved in 90% ethanol) for one minute. Finally, the tissues were washed three times with absolute alcohol and then transferred to xylene for cleaning the contrast [27].

The sections were mounted in Canada balsam by putting a drop of resin on a slide and placing the sections on the slides and photographed with digital camera attached to a compound microscope.

2.2.3. Anatomical Traits

For the collection of root samples, tertiary roots were exposed by digging in the rhizospheric soil of each plant. Soil digging was done up to 30 cm, and the thickest (tertiary) root was selected for anatomical sectioning of all traits. The root cross sectional area (mm²) was measured under a compound light microscope (Olympus MX63, Japan) by recording maximum length and width of the root sections. The exodermis thickness and cell area was measured (µm) randomly from three different sides following previously described methods [12,13]. The cortical cell area was measured using the length and width of selected cortical cells in µm². The sclerenchyma thickness and cell area was measured from the outer cortical region. The area of total vascular region was measured by recording the length and width of vascular regions in µm².

The length and width of selected metaxylem and phloem vessel regions were used to record metaxylem and phloem areas in µm². The general formula used to record the area of different root parts modified from the area of a circle (πr²) is as follows:

Area = (Maximum length × Maximum width/28) × 22

(2)

2.2.4. Statistical Analysis

There were nine fruits selected from each plant of eleven selected genotypes with five replications to record various fruit morphological and biochemical data. Root anatomical data were recorded for each three roots as replicates. The data were subjected to statistical software analysis (Statistics 8.1) under completely randomized design (CRD) to compare similarities in fruit morphological, biochemical, and root anatomical characters at a 5% probability for analysis of variance. Similarly, variation among mean values of genotypes for a specific studied character was compared using Tukey’s and multiple range tests.

Principal component analysis was performed on the data of fruit morpho-chemical and root anatomical observations of 11 selected pomegranate genotypes using XLSTAT (Software 2016) for biplot graphs and agglomerative hierarchical clustering.

3. Results

3.1. Meteorological Details of Pomegranate Sampling Regions

Meteorological data of rainfall, relative humidity (RH), and minimum and maximum temperatures from 2014 to 2018 showed a significant difference in five selected regions (Figure 2). The highest mean rainfall and relative humidity (RH) were recorded in Chakwal and Multan, respectively, while the minimum was recorded in Quetta. The highest average maximum and minimum temperatures were recorded in Bahawalpur and Quetta, respectively.

3.2. Fruit Morphology of Selected Pomegranate Genotypes and Their Relation with Them

The biplot differentiated pomegranate accessions into six major groups according to their morpho-biochemical traits (Figure 3). The commercial accession (Sava) had a strong positive correlation with total soluble solids (TSS), rind weight, and fruit weight; TG was strongly associated with vitamin C (Vit.C), aril weight, and fruit diameter; TK-I, TK-II, and QW-I had a strong correlation with titratable acidity (TA) and crown height (CH); however, all non-commercial accessions (CD-I, CD-II; KK-I, KK-II, and BW-G) had strong positive correlation with high wood portion index (WPI) and high seed weight, as shown in Figure 3. Moreover, the biplot showed high level of variations in selected genotypes for recorded observations as accessions were scattered in all four planes of the plot.

The highest fruit weight (373.97 g) was observed in the commercial genotype of Muzaffargarh (Sava) followed by TG (a commercial cultivar of Tarnab) with a fruit weight of 249.68 g as shown in

Table 2. Fruit morphological and biochemical traits of selected pomegranate accessions (p ≤ 0.05).

Genotypes	Fruit Wt. (g)	FD(mm)	CH (mm)	R. Wt. (g)	RT (mm)	Aril Wt. (g)	Seed Wt. (g)	WPI (%)	TSS (°Brix)	TA (%)	TS (%)	Vit. C (%)
TG	249.68 ± 44.23	72.12 ± 6.55	6.85 ± 0.99	72.23 ± 15.08	3.59 ± 0.49	118.01 ± 11.49	1.89 ± 0.31	0.98 ± 0.07	14.73 ± 0.47	0.52 ± 0.34	44.5 ± 0.4	0.63 ± 0.03
TK-I	218.88 ± 36.18	67.38 ± 5.61	16.13 ± 23.21	67.64 ± 7.45	3.50 ± 0.28	120.53 ± 11.10	3.57 ± 0.93	1.06 ± 0.03	14.03 ± 0.97	0.36 ± 0.07	48.33 ± 0.32	0.46 ± 0.02
TK-II	229.11 ± 32.29	70.99 ± 3.42	8.74 ± 1.69	62.73 ± 14.04	3.26 ± 0.69	47.90 ± 8.84	4.48 ± 0.33	1.01 ± 0.06	13.56 ± 0.47	0.17 ± 0.01	53.16 ± 0.5	0.47 ± 0.02
KK-I	60.94 ± 31.84	45.19 ± 8.98	10.67 ± 2.41	28.62 ± 10.71	3.31 ± 0.36	35.06 ± 7.11	1.48 ± 0.85	4.38 ± 0.52	14.56 ± 0.7	0.27 ± 0.05	39.71 ± 1.44	0.34 ± 0.11
KK-II	71.63 ± 21.04	49.21 ± 4.98	8.57 ± 1.05	27.06 ± 5.31	3.32 ± 0.47	28.17 ± 13.66	5.42 ± 0.77	3.98 ± 0.57	14.26 ± 1.91	0.15 ± 0.05	38.13 ± 0.68	0.39 ± 0.16
KK-III	203.955 ± 15.26	64.60 ± 7.99	8.53 ± 0.66	95.85 ± 10.58	5.25 ± 0.91	50.47 ± 17.54	3.88 ± 0.51	0.98 ± 0.08	14.29 ± 0.55	0.29 ± 0.02	36.47 ± 1.09	0.48 ± 0.15
CD-I	151.5 ± 25.79	53.36 ± 5.48	7.87 ± 0.48	53.31 ± 4.55	3.71 ± 0.33	49.38 ± 6.72	3.25 ± 0.31	1.06 ± 0.12	14.33 ± 0.82	0.13 ± 0.02	35.53 ± 0.30	0.39 ± 0.08
CD-II	135.38 ± 27.7	51.40 ± 5.8	8.59 ± 0.45	46.76 ± 3.88	3.65 ± 0.52	50.33 ± 9.36	4.13 ± 0.39	1.04 ± 0.07	12.73 ± 1.29	0.19 ± 0.01	33.9 ± 0.2	0.23 ± 0.08
QW-I	225.61 ± 34.32	75.92 ± 4.21	10.88 ± 3.37	54.62 ± 16.52	3.37 ± 0.94	16.86 ± 2.81	3.11 ± 1.13	0.93 ± 0.06	15.33 ± 0.58	1.04 ± 0.14	45.37 ± 0.30	0.68 ± 0.04
BW-G	121.84 ± 14.74	57.29 ± 3.69	8.09 ± 0.74	34.54 ± 2.2	1.89 ± 0.22	36.18 ± 2.0	3.51 ± 0.32	1.01 ± 0.03	11.37 ± 0.73	0.35 ± 0.04	43.97 ± 2.66	0.21 ± 0.01
Sava	373.97 ± 28.62	86.18 ± 1.85	7.47 ± 0.38	97.64 ± 9.17	3.14 ± 0.16	74.82 ± 3.95	2.74 ± 0.58	0.94 ± 0.02	16.46 ± 0.65	0.09 ± 0.005	38.57 ± 1.10	0.51 ± 0.02

FD: Fruit diameter, CH: Crown height, R. wt.: Rind weight, RT: Rind thickness, WPI: Wood portion index, TA: Titratable acidity, TS: Total sugars; Values are means ± SD. Mean values in bold style were highly significantly different in the respective genotypes.

. In earlier studies, both cultivars had a high fruit diameter with a sweeter taste. The lowest fruit weight (60.94 g and 71.63 g) was recorded in red and white pink genotypes, respectively, of Kallar-kahar and Chakwal (Salt range of Potuhar) with a sour aril taste; however, genotypes of Agriculture Research Institute Tarnab (TK-I, TK-II) and Quetta (QW-I) had ≥200 g fruit weight.

High rind weight (97.64 g) was recorded in coefficient of variance (cv) Sava followed by KK-III and TG with rind weights of 95.85 g and 72.23 g, respectively; however, high rind thickness (5.25 mm) was measured in the non-commercial genotype (KK-III) of Kanhatti (Table 2). The maximum 100 aril weight (120.53 g) was recorded in TK-I and TG; however, 100 seed weight was at a maximum (5.42g) in KK-II. The wood portion index (WPI) was high (4.38%) in cv. KK-I with the minimum value (0.93%) in QW-I and other commercial genotypes showed high juiciness. The highest total soluble solids (16.46 °Brix) were recorded in Sava followed by QW-I and TG; however, titratable acidity was also high in these genotypes.

Total sugar contents were recorded at a maximum (53.16% and 48.33%) in TK-II and TK-I, respectively. Ascorbic acid concentrations were high in commercial accession (QW-I) followed by TG and Sava, as shown in Table 2.

3.3. Variation in Root Anatomical Structure of Selected Pomegranate Genotypes and Their Relation

There was a significant difference among all genotypes in the root cross-sectional area as detailed in

Table 3. Root anatomical variation in selected pomegranate accessions of different regions.

Geno-Types	Root Area (µm2)	Epidermis Area (µm2)	Cortex Thickness (µm)	Cortex Area (µm2)	Vascular Area (µm2)	Phloem Area (µm2)	Metaxylem Area (µm2)
TG	176405.14 ± 1271.66 c	91.23 ± 1.09 a	91.14 ± 2.17 ab	233.83 ± 2.96 d	81612.52 ± 155.41 e	2066.06 ± 31.08 c	514.11 ± 83.69 cd
TK-I	178594.23 ± 7047.32 bc	79.86 ± 10.15 b	74.50 ± 3.31 bcd	301.59 ± 18.82 b	95721.29 ± 604.38 b	1412.48 ± 22.45	587.08 ± 33.44 bc
TK-II	185651.07 ± 2128.27 a	56.78 ± 3.0 c	86.80 ± 2.17 abc	223.22 ± 8.74 d	95897.64 ± 930.79 b	2005.32 ± 128.38 cd	632.06 ± 71.66 ab
KK-I	170722.18 ± 1673.90 d	46.89 ± 3.70 d	71.61 ± 2.17 cd	65.99 ± 2.46 f	77187.50 ± 945.31 f	2324.99 ± 149.52 b	139.36 ± 34.38 g
KK-II	157151.13 ± 3846.11 f	57.27 ± 3.33 c	104.74 ± 2.39 a	462.74 ± 22.99 a	65267.82 ± 1321.69 i	2240.88 ± 57.83 b	492.33 ± 85.10 d
KK-III	163550.63 ± 2401.13 e	32.56 ± 1.28 e	73.99 ± 1.95 bcd	129.81 ± 2.13 e	114382.10 ± 1190.97 a	1894.82 ± 139.02 d	205.66 ± 23.38 fg
CD-I	154245.51 ± 793.01 f	40.69 ± 6.41 de	27.49 ± 36.33 e	90.03 ± 5.95 f	84711.76 ± 256.76 d	1501.40 ± 97.28 e	566.08 ± 57.44 bcd
CD-II	157847.44 ± 1325.46 f	35.52 ± 4.0 e	66.55 ± 2.51 d	90.03 ± 14.95 f	88262.39 ± 459.65 c	2037.88 ± 131.93 cd	277.62 ± 20.81 f
QW-I	156176.83 ± 1482.98 f	40.69 ± 6.41 de	73.20 ± 3.08 bcd	267.62 ± 23.79 c	85905.58 ± 929.22 d	2369.14 ± 61.95 b	376.64 ± 19.90 e
BP-G	182276.81 ± 1469.34 ab	21.73 ± 1.57 f	86.87 ± 0.12 abc	131.34 ± 33.96 e	74473.05 ± 368.97 g	1389.91 ± 76.93 e	515.51 ± 53.53 cd
Sava	166240.43 ± 2958.74 de	97.43 ± 7.70 a	78.92 ± 3.22 bcd	304.62 ± 22.77 b	71902.88 ± 908.30 h	2730.48 ± 115.35 a	717.77 ± 55.87 a

Values are Mean ± SD (n = 9); Small superscript letters represent significant differences (p < 0.05) among treatment means.

and Figure 4. The maximum root cross-sectional area (185,651 µm²) was recorded in accession TK-II, which was statistically on par with BP-G. Pomegranate accessions KK-II, CD-I, CD-II, and QW-I had a statistically similar low root cross-sectional area. High root epidermis area (97.43 µm²) was recorded in accession Sava, which was statistically on par with commercial accession TG; however, all non-commercial genotypes had less epidermis area, except QW-I, which is a commercial accession (Table 3). Root cortical thickness was significantly high (104.73 µm²) in Chakwal accession KK-II, which was statistically on par with accessions TG, TK-II, and BP-G. The accession KK-II yielded a maximum cortical cell area (462.74 µm²) followed by Sava (304.62 µm²). Similarly, significant (p < 0.05) differences were found in selected accessions regarding the root vascular area. The accessions KK-III had a maximum vascular area (114,382.10 µm²) followed by TK-1 and TK-II (Table 3). The maximum phloem area (2730.48 µm²) was recorded in accession Sava followed by QW-I, KK-I, and KK-II. The metaxylem vessel area varied significantly (p < 0.05) in all selected pomegranate accessions. The metaxylem vessel area was at a maximum (717.77 µm²) in accession Sava, which was statistically on par with TK-II (632.06 µm²). The biplot analysis of root anatomical data of selected pomegranate genotypes showed a successful scattering of all genotypes in four planes of the plot, which proved the diverse nature of root anatomy and strong association of these genotypes for various cross-sections of roots (Figure 5).

Accessions Sava and KK-II had strong positive associations with cortex area (CorA) and phloem area (PhlA), respectively; however, TG had a strong positive correlation with metaxylem area (MVA), epidermis area (EpA), and cordex thickness (CorT) of root sections; moreover, TK-I and TK-II had a strong association with root area (RA). Non-commercial accessions (CD-1 and II; KK-1and III) and one commercial accession (QW-I) gathered in same plan showing narrow diverse root anatomy in these accessions as shown in Figure 5.

3.4. Clustering of Pomegranate Genotypes Based on Fruit Morpho-Chemical and Root Anatomical Studies of Selected Pomegranate Accessions

Pomegranate accessions were successfully clustered into three main classes based on various morphological (fruit size, aerial characters, juiciness, total soluble solids (TSS), acidity, and ascorbic acid contents) and root anatomical characters (epidermal, vascular, cortex, and metaxylem areas). There was a 60.16% variation among and 39.84% within classes (Figure 6). Eight genotypes of diverse regions were clustered into class 1, which was further divided into two subclasses (Figure 6). Two genotypes of Chakwal (CD-I and CD-II) were successfully grouped with one commercial genotype of Quetta (QW-I), proving high similarity (C-Ia). In subclass C-Ib, five accessions were clustered based on various morphological and anatomical traits. Non-commercial accessions of Bahawalpur (BP-G) and Khushab Kanhati garden (KK-I and KK-II) were grouped with commercial accessions of Muzaffargarh (Sava) and Peshawar (TG) districts. However, the two accessions of district Peshawar (Tk-I & TK-II) grouped in class C-II and were closely related to Khushab accession KK-III (Figure 6).

4. Discussion

A repository of pomegranate germplasm collected from various regions of Pakistan is established at the University of Agriculture, Faisalabad, Pakistan, to conserve crop genetic resources. The exploitation of germplasm diversity based on morphological, biochemical, physiological, and genetic traits has significance in germplasm release and breeding programs. In this study, we have measured the phenotypic and biochemical diversity and mapped anatomical adaptations in pomegranate under diverse climatic regions.

Morphological variations in pomegranate fruits were observed in selected accessions, which are endorsed by previous findings of Hasnaoui et al. [28] and Nafees et al. [20], who reported variations in wild and cultivated pomegranates including high fruit weight with less WPI. High fruit size and rind weight, aril weight, and juice contents are found in the cultivated pomegranates of Tunisia and Turkey [6,29]. A high TSS variation was also recorded in Tunisian, Spanish and Persian pomegranate cultivars (14.33 to 16.30 °Brix) [29,30,31], respectively, while, ≥19 °Brix TSS was recorded by Zarei et al. [32] in Iranian pomegranates, which is higher than what was recorded in our accessions. Iranian pomegranate fruit juice contains 9.68–17.45 mg/100 mL Vitamin C content [33], which is many folds higher than our studied pomegranate accessions; however, various Pakistani wild and commercial pomegranate accessions had various organic acids and sugars [34].

High variation among various root anatomical parameters in pomegranate germplasm proved its adaptation under cold temperate to tropical, subtropical, and desert zones. This has also been reported by researchers [12,13] who stated that various ecological influences result in structural changes in root tissues, which increases the chances of adaptation and commercialization. Hence, various root characteristics could be used in germplasm characterization and pomegranate breeding programs [10,11,35]. In this study, roots emerged from stem cuttings under Faisalabad conditions showed diverse anatomy, which may be due to the stem cuttings having been collected from plants growing under diverse agro-climatic regions. Moreover, these genotypes had adopted that specific ecological condition and reflected the same results under uniform agro-climatic conditions of Faisalabad district. No abiotic stress like drought, salinity, or soil-borne diseases was observed, which could impact pomegranate root anatomy.

Overall, the root anatomy studied in accessions varied significantly; however, diverse anatomy was also observed in accessions growing in the same district/region, which might be due to variations in their genetic makeup. Thick-walled and diverse epidermis without exodermis in the roots of selected pomegranate genotypes ensured their adaptability in a diverse climate. This is corroborated by previous studies [33,34,35,36] that stated that a high level of variation in root exodermis and endoderm composition in plant species is due to lignin deposition dependent on the diverse agro-climate of the regions. The small metaxylem vessels observed in studied accessions are associated with high survival potential as reported previously [37]. However, in contrast, diverse agro-climatic conditions reduced the xylem vessel area and affected plant physiology and its biomass [17,38]. Moreover, the better adaptation of various asparagus genotypes in diverse environments is due to modified root structures, so root anatomical properties are good indicators of habitat ecology and can effectively be used as markers for stress tolerance in plant species. Different evolutionary routes for date palm cultivars are due to significant variations in the root structures [11]. This high variation of fruit and root parameters in selected pomegranate genotypes might be due to a high variation in the climatic conditions of their growing regions, and this was also recorded in this study, supported by [38], which stated that the various morpho-biochemical and other physiological factors are directly linked with climatic conditions of the regions.

The studied pomegranate accessions successfully clustered based on various fruit morphological and root anatomical traits, which shows that, regardless of growing regions, most of these germplasms share their genetic makeup and gene flow from one region to another. Similar results have been reported by previous studies [20,38], where 115 wild and domesticated pomegranates were successfully clustered based on morpho-molecular and biochemical analyses. Moreover, Turkish pomegranate genotypes also clustered based on similarities of morpho-chemical characters of cultivars regardless of growing regions.

5. Conclusions

It is concluded from this study that successful commercial pomegranate production from temperate to subtropical and arid zones of Pakistan might be due to the high variation in root anatomical characteristics. Similar root characteristics of commercial genotypes of Peshawar (TG, TK-I, TK-II), Quetta (QW), and Muzaffargarh (Sava) proved their potential for extension and cultivation in different regions. However, this needs the acclimatization and optimization of production technology in order to open new horizons of pomegranate production in Pakistan, and likewise around the globe, to address malnutrition and food security issues in the region.