Tendril Anatomy: A Tool for Correct Identification among Cucurbitaceous Taxa

Abstract

This research examined the histological micro-structure of tendril vasculature in cucurbitaceous taxa. In this research, the tendril anatomy of 17 taxa of Cucurbitaceae categorized into seven genera, including Cucumis (five species), Cucurbita and Luffa (three species each), Citrullus and Momordica (two species each) while Lagenaria and Praecitrullus (one species each), collected from different areas of the Thal desert were examined via microscopic imaging to explore its taxonomic significance. Tendril transverse sections were cut with a Shandon Microtome to prepare slides. The distinctive characteristics of taxonomic value (qualitative and quantitative) include tendril and vascular bundle shape, variation in the number of vascular bundles, tendril diameter length, layers of sclerenchyma, and shape of collenchyma and epidermal cells. Tendril shapes observed are irregular, slightly oval-shaped, slightly C shaped, angular (4-angled, 6-angled, or polygonal), and star shaped. Quantitative measurements were taken to analyze the data statistically using SPSS software. Cucurbita pepo had a maximum tendril diameter length of 656.1 µm and a minimum in Momordica balsamina of 123.05 µm. The highest number of vascular bundles (12) were noticed in Luffa acutangula var.amara. Angular type was prominent in collenchyma, and irregular shape was dominant in sclerenchyma cells. A maximum of seven to nine sclerenchyma layers were present in Lagenaria siceraria and a minimum of two or three layers in Cucumis melo subsp. agrestis, Cucumis melo var. flexuosus, and Cucumis melo var.cantalupensis. Epidermis cells also show great variations with a rectangular shape being dominant. Statistical UPGMA dendrogram clustering of tendril vasculature traits shows that histological sections studied with microscopic techniques can be used to identify species and will play a vital role in future taxonomic and phylogenic linkages.

Full text

Plants 2022, 11, 3273. https://doi.org/10.3390/plants11233273 www.mdpi.com/journal/plants
Article
Tendril Anatomy: A Tool for Correct Identification among
Cucurbitaceous taxa
Naveed Abbas 1, Muhammad Zafar 1,*, Mushtaq Ahmad 1,2, Ashwaq T. Althobaiti 3, Mohamed Fawzy Ramadan 4,
Trobjon Makhkamov 5, Yusufjon Gafforov 6,7, Khislat Khaydarov 8, Muhammad Kabir 9, Shazia Sultana 1,
Salman Majeed 1,10 and Tajalla Batool 1
1 Department of Plant Systematics, Biodiversity Lab, Quaid-i-Azam University,
Islamabad 45320, Pakistan
2 Pakistan Academy of Sciences Islamabad, Islamabad 46000, Pakistan
3 Department of Biology, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
4 Department of Clinical Nutrition, Faculty of Applied Medical Sciences, Umm Al-Qura University,
Makkah 21961, Saudi Arabia
5 Department of Forestry and landscape Design, Tashkent State Agrarian University, 2 A., Universitet Str.,
Kibray District, Tashkent 100700, Uzbekistan
6 Mycology Laboratory, Institute of Botany, Academy of Sciences of Republic of Uzbekistan, 32 Durmon Yuli,
Tashkent 100125, Uzbekistan
7 AKFA University, 264 Milliy Bog Street, Tashkent 111221, Uzbekistan
8 Faculty of Biology, Samarkand State University, Uzbekistan, Universitetsty Bulvvar Street-15,
Samarkand 140104, Uzbekistan
9 Department of Biological Sciences Ex University of Sargodha sub Campus Bhakkr, Thal University Bhakkar,
Bhakkar 30000, Pakistan
10 Department of Botany, University of Mianwali, Mianwali 42200, Pakistan
* Correspondence: zafar@qau.edu.pk; Tel.: +92-05190643149
Abstract: This research examined the histological micro-structure of tendril vasculature in cucurbi-
taceous taxa. In this research, the tendril anatomy of 17 taxa of Cucurbitaceae categorized into seven
genera, including Cucumis (five species), Cucurbita and Luffa (three species each), Citrullus and
Momordica (two species each) while Lagenaria and Praecitrullus (one species each), collected from
different areas of the Thal desert were examined via microscopic imaging to explore its taxonomic
significance. Tendril transverse sections were cut with a Shandon Microtome to prepare slides. The
distinctive characteristics of taxonomic value (qualitative and quantitative) include tendril and vas-
cular bundle shape, variation in the number of vascular bundles, tendril diameter length, layers of
sclerenchyma, and shape of collenchyma and epidermal cells. Tendril shapes observed are irregu-
lar, slightly oval-shaped, slightly C shaped, angular (4-angled, 6-angled, or polygonal), and star
shaped. Quantitative measurements were taken to analyze the data statistically using SPSS soft-
ware. Cucurbita pepo had a maximum tendril diameter length of 656.1 µm and a minimum in Momor-
dica balsamina of 123.05 µm. The highest number of vascular bundles (12) were noticed in Luffa
acutangula var.amara. Angular type was prominent in collenchyma, and irregular shape was domi-
nant in sclerenchyma cells. A maximum of seven to nine sclerenchyma layers were present in Lage-
naria siceraria and a minimum of two or three layers in Cucumis melo subsp. agrestis, Cucumis melo
var. flexuosus, and Cucumis melo var.cantalupensis. Epidermis cells also show great variations with a
rectangular shape being dominant. Statistical UPGMA dendrogram clustering of tendril vascula-
ture traits shows that histological sections studied with microscopic techniques can be used to iden-
tify species and will play a vital role in future taxonomic and phylogenic linkages.
Keywords: anatomy; Cucurbitaceae; micromorphology; parenchyma; vessel elements
Citation:
Abbas, N.; Zafar, M.;
Ahmad, M.; Althobaiti, A.T.;
Ramadan, M.F.; Makhkamov, T.K.;
Gafforov, Y.; Kudratovich, K.K.;
Kabir, M.;
Sultana, S.; et al. Tendril
Anatomy: A Tool for Correct
Identification among
Cucurbitaceous taxa.
Plants 2022, 11,
3273. https://doi.org/
10.3390/
plants11233273
Academic Editors: Ioannis Bazos,
Kostas Kougioumoutzis and
Panayotis Dimopoulos
Received: 16 October 2022
Accepted: 28 October 2022
Published: 28 November 2022
Publisher’s Note:
MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and institu-
tional affiliations.
Copyright:
© 2022 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).

Plants 2022, 11, 3273 2 of 29
1. Introduction
The Cucurbitaceae family, or cucurbits, are most widely distributed in subtropical
and tropical climates, with hotspots in West Africa, Southeast Asia, Mexico, and Mada-
gascar [1]. Cucurbitaceous members (watermelons, cucumbers, luffas, pumpkin, cour-
gettes, zucchini, and summer squash) are all edible and can be found growing across all
continents. Around 800 species and 130 genera can be distributed worldwide [2]. In West
Africa, this family is represented by 24 genera and 54 species [3]. In Pakistan, it has 33
species across 17 genera, including both domesticated (22 species) and wild species (11
species) [4]. The wild genera are Coccinia, Lageneria, Luffa, Momordica, and Zehneria,
whereas cultivated include Citrullus, Cucumis, Cucurbita, Cucumeropsis, Lagenaria, Telfairia,
and Trichosanthes [4]. However, Pakistan’s cucurbit species with high nutritious potential
remain unexplored [5].
Citrullus lanatus is a succulent species belonging to the Citrullus genus and are desert
vines and the only genus in the family with pinnatifid leaves. Watermelon (three varieties)
and brown-seeded melon, both with bitter pulp, are members with solitary staminate
flowers, tiny sepals, a basal corolla sectioned into five parts, and fleshy fruits. These are
members of the subspecies C. lanatus, which is commonly cultivated in Pakistan. Cucumis,
true melons, honey melons, and West Indian gherkins are all members of the twinning,
tendril-bearing plants that belong to the Cucumis genus [6]. The leaves rarely split beyond
the center. The fruits are smooth, green-lined, or hairy, with the appearance of a ground
trailer. Cucurbita is a genus with approximately 20 species. The primary cultivated squash
and pumpkin are four different Cucurbita species: C. pepo, C. sativus, C. maxima, and C. pepo
var cylindrica. Ripe and immature fruit is the most important edible plant parts, although
some species also consume seeds, flowers, roots, and even leaves. Cucurbitaceous species
are not the only source of food, but are also used as a nutraceutical and pharmacothera-
peutic potential [7]. The pepo’s sweet, delicately flavored, juicy flesh is eaten raw, fre-
quently as a dessert. Cucumis melo, often known as sweet melon, is a fruit, not a vegetable.
Cucurbitaceous family members have lianous plant bodies, peculiar fleshy fruits
(known as pepo), and a similar system of sex determination. The Cucurbitaceous species
are primarily herbaceous plants with diverse pubescence and tuberous roots [2,8]. They
are also physically distinguished by frequently angled stems and bicollateral vascular
bundles that are frequently grouped in two concentric rings. The leaves are petiolate, ex-
stipulate, alternating, often palmately veined, simple, or sedately complex, with extra-flo-
ral nectaries. The tendrils are lateral to the base of the petiole, usually one to four at each
node, branching, simple-lobed, with a non-spiraling base [4].
Tendrils are an excellent example of convergent evolution because they have evolved
numerous times among angiosperms. They can be found in lineages that are not closely
related, such as Magnoliales and Asterales [9]. Tendrils are a perfect demonstration of
their wide variation in morphology and ontogeny over the course of evolution. Tendrils
can develop from modified twigs, pedicel, stipules, entire leaves, leaflets, leaf bracts, leaf
apex, and inflorescences [10].
Environmental changes do not affect a plant’s anatomical characteristics [11]. Ana-
tomical information was utilized to identify plant species, genera, and families. It is fre-
quently employed in systematic identification, giving anomalous groupings a better clas-
sification position and illuminating relationship patterns that morphological traits may
not have entirely conveyed. Nevertheless, it has been documented that cucurbit species in
Pakistan can be distinguished by their plant morphology and anatomy [3]. Several [12–
14] researchers describe anatomical characters of Cucurbitaceous species however, tendril
micromorphology is not visualized [15]. Plant cell and tissue distributions such as scle-
renchyma, vascular bundles, and other anatomic traits have been described at various
systematic levels for species delimitation [16]. The presence of trichomes on the lower sur-
face of the leaves, which reflects significant taxonomic relevance, is well recognized in the
Cucurbitaceae [17]. Comparative and systematic studies on the anatomy of the various

Plants 2022, 11, 3273 3 of 29
vegetative organs (root, stem, and leaf) of the species of the Cucurbitaceae family were
carried out [13,14,18,19].
Thus, to better understand the systematic relationships, there is a need to study dif-
ferent field characteristics, like the anatomical features of tendrils involved in plant tax-
onomy. This study aimed to analyze and describe the significant tendril micromorpho-
logical traits which will contribute to the anatomy of the Cucurbitaceae and provide fea-
tures for accurate species identification and their taxonomic implications.
2. Results
Tendril anatomical traits observed in the current investigation were outline, vascu-
larization forms, vessel elements, collenchyma, chlorenchyma, sclerenchyma, as well as
parenchyma tissues as illustrated (Figure 1–9). The summarized qualitative and quantita-
tive results are given in (Tables 1–4). Observing their recorded taxonomic evaluation, cu-
curbitaceous taxa showed significant size and shape variation (Figure 1–9).

Plants 2022, 11, 3273 4 of 29
Table 1. Qualitative tendril anatomical characters of Cucurbitaceous species.
Sr No.
Cucurbitaceous Taxa
Epidermal Cell
Shape
Collenchyma
Cell Type
Chlorenchyma
Cell Shape
Sclerenchyma
Cell Shape
Parenchyma
Cell Shape
Vascular
Bundle Shape
Vascular
Bundles
Tendril Outline in
Transverse View
1.
Citrullus colocynthis (L) Schrad.
Rectangular and
isodiametric
Angular
Rectangular to
irregular
Tetragonal to
polygonal
Polygonal Oval Subsidiary Slightly oval
2.
Citrullus lanatus (Thunb.)
Matsum.& Nakai
Rectangular Angular Irregular
Polygonal to
irregular
Irregular Elliptical Subsidiary
Irregular, slightly v
shaped
3.
Cucumis melo L.
Rectangular
Angular
Irregular
Irregular
Irregular
Irregular
Subsidiary
Irregular shaped
4.
Cucumis melo subsp. agrestis
(Naudin) Pangalo
Oval to irregular Angular Irregular Polygonal Polygonal Irregular Subsidiary 4 angled
5.
Cucumis melo var. flexuosus (L.)
Naudin
Rectangular, square,
and isodiametric
Angular Irregular
Tetragonal to
polygonal
Polygonal Irregular Subsidiary Irregular shaped
6.
Cucumis melo var.cantalupensis
Naudin
Rectangular to
square
Angular Polygonal
Triangular to
polygonal
Irregular Irregular Central Irregular shaped
7.
Cucumis sativus L. Rectangular Angular Rectangular
Tetragonal to
polygonal
Irregular
Elliptical and
irregular
Subsidiary Star-shaped
8.
Cucurbita maxima Duchesne Rectangular Angular
Rectangular to
polygonal
Irregular Irregular
Dumbbell and
irregular
Subsidiary Irregular shaped
9.
Cucurbita pepo L.
Rectangular to
square
Lamellar and
angular
Polygonal Irregular Irregular Irregular Subsidiary Six angled
10.
Cucurbita pepo var. cylindrica
Rectangular, square
to polygonal
Lamellar and
angular
Polygonal Irregular Irregular
Oval and
irregular
Subsidiary Slightly c shaped
11.
Lagenaria siceraria (Molina)
Standl.
Rectangular Lamellar
Rectangular to
irregular
Irregular Irregular Rounded Subsidiary Irregular with hollow pith
12.
Luffa acutangula (L.) Roxb.
Irregular
Angular
Irregular
Irregular
Irregular
Rounded
Subsidiary
Irregular with hollow pith
13.
Luffa acutangula var. amara
C.B.Clarke
Rectangular, square
to polygonal
Angular Polygonal
Polygonal and
irregular
Irregular
Round and
oval
Subsidiary Irregular polygonal
14.
Luffa cylindrica (L) M.Roem
Rectangular to
square
Angular
Hexagonal to
polygonal
Tetragonal to
polygonal
Irregular
Round and
elliptical
Subsidiary Six angled
15.
Momordica charantia L.
Rectangular to
irregular
Angular
Pentagonal to
polygonal
Irregular Irregular
Rounded and
elliptical
Subsidiary 4 angled
16
Momordica balsamina L.
Pentagonal to
hexagonal Angular
Rectangular to
polygonal
Tetragonal to
hexagonal
Pentagonal to
irregular
Oval and
irregular Central 4 angled

Plants 2022, 11, 3273 5 of 29
17
Praecitrullus fistulosus (Stocks)
Pangalo
Rectangular Angular
Pentagonal to
polygonal
Irregular Irregular Irregular Subsidiary
Polygonal, slightly U
shaped
Table 2. Quantitative analysis of tendril anatomy of Cucurbitaceous taxa.
Sr No.
Cucurbitaceous Taxa
No. Of Vascular Bundles Epidermal
Cell Layer
Collenchyma
Cell Layer
Chlorenchyma
Cell Layer
Sclerenchyma
Cell Layer
Parenchyma Cell
Layer
Vessel
Elements
1.
Citrullus colocynthis (L) Schrad.
4
1
3–4
1–2
3–5
4
6
2.
Citrullus lanatus (Thunb.) Matsum.& Nakai
6
1
2–3
1
3–4
4
5
3.
Cucumis melo L.
5
1
3
1
3
4
8
4.
Cucumis melo subsp. agrestis (Naudin) Pangalo
5
1
3
1–2
2–3
4
8
5.
Cucumis melo var. flexuosus (L.) Naudin
7
1
3
2
2–3
3
5
6.
Cucumis melo var.cantalupensis Naudin
6
1
3
2
2–3
4
4
7.
Cucumis sativus L.
5
1
4
1
2–4
6
15
8.
Cucurbita maxima Duchesne
7
1
3
2
5
4
9
9.
Cucurbita pepo L.
6
1
6
2
3–4
4
7
10.
Cucurbita pepo var. cylindrica
7
1
4
2
3–4
3
9
11.
Lagenaria siceraria (Molina) Standl.
7
1
3–4
2–3
7–9
5
8
12.
Luffa acutangula (L.) Roxb.
8
1
5
1
6–8
3
8
13.
Luffa acutangula var.amara C.B.Clarke
12
1
6
2
6
2
9
14.
Luffa cylindrica (L) M.Roem
8
1
5
1
6
3
9
15.
Momordica charantia L.
7
1
5
1
4
3
9
16
Momordica balsamina L.
3
1
3–5
1
1
3–4
10
17
Praecitrullus fistulosus (Stocks) Pangalo
10
1
2–5
2
4
5
5
Table 3. Quantitative tendril anatomical data of Cucurbitaceous species.
Sr
No.
Cucurbitaceous taxa
L x W
Tendril Diameter (µm)
Upper
Epidermal Cell (µm)
Lower
Epidermis Cell (µm)
Collenchyma
Cell (µm)
1.
Citrullus colocynthis (L)
Schrad.
L
314 − 326.25 = 321.65 ± 5.02
16.75 − 23.25 = 19.85 ± 2.57
16.75 − 22.50 = 19.65 ± 2.25
10.50 − 21.25 = 16.80 ± 4.74
W
297 − 304.75 = 301.10 ± 2.77
12.25 − 17.75 = 14.95 ± 2.13
12.75 − 18 = 15.10 ± 2.19
4.25 − 16.75 = 11 ± 4.72
2.
Citrullus lanatus (Thunb.)
Matsum.& Nakai
L
512.50 − 517.75 = 514.95 ±
2.18
15.75
− 25.25 = 20.90 ± 3.75
14.75
− 25.75 = 20.55 ± 4.93
16.25
− 23.25 = 19.80 ± 2.56
W
250 − 258.75 = 253.50 ± 3.24
10 − 20 = 12.9 ± 4.14
10.25 − 20.50 = 13.55 ± 4.12
7.50 − 12.50 = 10.05 ± 1.97
3.
Cucumis melo
L.
L
126.75 − 145.25 = 134.10 ±
7.44
10.50
− 21.75 = 16.20 ± 4.50833
10
− 20.75 = 15.55 ± 4.28442
12.00
− 21.25 = 16.20 ± 3.77

Plants 2022, 11, 3273 6 of 29
W
122.75 − 138 = 130 ± 6.38
8.75 − 13.75 = 11.05 ± 2.07
8.75 − 13 = 10.6 ± 1.85
7.50 − 13.75 = 10.65 ± 2.75
4.
Cucumis melo subsp.
agrestis
(Naudin) Pangalo
L
200.50 − 212.50 = 205.35 ± 5
3.75 − 25.50 = 16.70 ± 9.13
4.75 − 25.50 = 12.85 ± 9.49
13.00 − 35.50 = 25.50 ± 9.78
W
183.50 − 188.50 = 186.30 ±
2.13
3.75
− 18 = 11.55 ± 6.18
2.50
− 15.25 = 7.25 ± 4.97
8.25
− 25.50 = 15.45 ± 6.54
5.
Cucumis melo var. flexuosus
(L.) Naudin
L
325.25 − 333 = 328.85 ± 3.18
13 − 50.25 = 23.15 ± 15.28
13.50 − 45.25 = 22.50 ± 12.90
11 − 28 = 18.50 ± 6.98
W
250.25 − 267.75 = 257.30 ±
7.73
7.75
− 16.75 = 11.15 ± 3.81
8
− 14.25 = 10.75 ± 2.97
2.75
− 13.25 = 8.5 ± 3.83813
6.
Cucumis melo
var
.cantalupensis Naudin
L
250.50 − 256.50 = 253.75 ±
2.384
18.75
− 26.25 = 22.7500 ± ± 2.87
19.50 − 26.50 = 22.6500 ±
2.831
13.00
− 24.75 = 18.7000 ± 4.396
W
225 − 231.25 = 227.60 ± 2.36
13.75 − 23.25 = 18.20 ± 3.40221
13.25 − 19.50 = 16.70 ± 2.558
12 − 14.25 = 13.1500 ± .91
7.
Cucumis sativus
L.
L
524.75 − 531.25 = 527.40 ±
2.71
20.75
− 30.00 = 26.0500 ± 4.052
17.75 − 30.50 = 25.9500 ±
5.7047
23.25
− 33.50 = 27.8500 ± 3.7358
W
225.00 − 238.25 = 229.40 ±
5.375
10.25
− 13.00 = 11.5500 ± 1.242
10.25 − 13.00 = 11.5500 ±
1.267
13.75
− 21.25 = 17.7000 ± 2.808
8.
Cucurbita maxima
Duchesne
L
589.75 − 600.25 = 595.60 ±
3.9115
19.25
− 42.25 = 27.3000 ± 10.24
19.50 − 41.00 = 27.2000 ±
9.8256
12.25
− 20.00 = 16.8500 ± 3.5820
W
486.25 − 494.75 = 489.60 ±
3.3241
9.25
− 20.00 = 14.2500 ± 4.10
9.75 − 19.75 = 14.4000 ±
3.91152
8.75
− 13.25 = 10.7000 ± 1.68077
9.
Cucurbita pepo
L.
L
650.00 − 662.75 = 656.10 ±
4.735
8.75
− 19.75 = 13.9000 ± 4.211
8.00 − 20.00 = 13.5000 ±
4.53114
9.50
− 15.25 = 12.8500 ± 2.29538
W
315.50 − 321.25 = 317.60 ±
2.1837
7.50
− 17.50 = 11.4500 ± 3.8786
7.25
− 18.00 = 11.3000 ±
4.298
7.25
− 14.75 = 10.4500 ± 3.0893
10.
Cucurbita pepo var.
cylindrica
L
361.25 − 368.00 = 364.45 ±
2.6774
22.50
− 31.25 = 26.5000 ± 3.579
22.75 − 31.00 = 26.5500 ±
3.188
16.75
− 25.00 = 21.7500 ± 3.292
W
162.75 − 170.25 = 1.67.00 ±
2.93
13.00
− 22.50 = 18.0500 ± 3.692
13.25 − 22.00 = 18.0000 ±
3.592
10.50
− 19.25 = 14.8500 ± 3.223
11.
Lagenaria siceraria
(Molina)
Standl.
L
471.25 − 487.75 = 477.85 ±
6.125
14.50
− 27.50 = 20.4500 ± 5.874
14.25 − 27.75 = 20.7000 ±
6.2759
22.50
− 30.50 = 27.7500 ± 3.172
W
210.00 − 223.75 = 217.65 ±
5.641
9.75
− 13.00 = 11.1000 ± 1.526
9.50 − 13.00 = 11.1500 ±
1.61632
8.75
− 12.75 = 10.7000 ± 1.71756
12.
Luffa acutangula
(L.) Roxb.
L
250 − 676.25 = 582.85 ± 186.37
8.50 − 20.50 = 15.05 ± 4.56002
7.25 − 20.50 = 14.7 ± 5.03550
13.00 − 28.50 = 21.20 ± 6.13
W
251.75 − 270.25 = 258.85 ±
7.86
5.25
− 10.50 = 8.05 ± 1.89
5.00
− 10.25 = 7.75 ± 1.87
7.50
− 17.50 = 12.20 ± 3.7
13.
Luffa acutangula var.amara
C.B.Clarke
L
451.25 − 480.50 = 472.45 ±
11.99
17.50
− 26 = 21.05 ± 4.19
17.75
− 27.50 = 21.3 ± 4.8
15.50
− 25 = 20.30 ± 3.83

Plants 2022, 11, 3273 7 of 29
W
219.75 − 252.75 = 229.20 ±
13.64
12.50
− 17 = 14.4 ± 1.82
12.75
− 16.25 = 4.1 ± 1.47479
12.75
− 22.75 = 15.9500 ± 3.98
14.
Luffa cylindrica
(L)
M.Roem
L
401.25 − 413.75 = 406.2 ±
4.84897
11.25
− 15.75 = 13.0000 ± 1.677
11.50 − 15.75 = 13.0000 ±
1.6488
15.75
− 23.25 = 19.7000 ± 3.2948
W
172.25 − 180.25 = 175.75 ±
2.915
8.00
− 15.25 = 11.6500 ± 2.637
8.25 − 15.25 = 11.7000 ±
2.51496
13.00
− 18.00 = 15.0500 ± 2.041
15.
Momordica charantia
L.
L
350.00 − 357.75 = 353.25 ±
3.177
11.75
− 17.00 = 14.7500 ± 2.186
12.00 − 17.00 = 14.6000 ±
2.2262
16.25
− 20.00 = 18.2500 ± 1.3578
W
160.75 − 167.25 = 164.55 ±
2.7064
8.75
− 11.00 = 9.9000 ± .96177
9.00
− 10.75 = 9.9500 ±
.71589
9.25
− 11.75 = 10.5500 ± 1.10962
16
Momordica balsamina
L.
L
122.25 − 124.0 = 123.05
± .81777
8.75
− 16.25 = 13.3000 ± 2.9811
8.50 − 16.00 = 13.3000 ±
3.15436
14.00
− 25.50 = 19.4500 ± 5.5884
W
112.25 − 114.00 = 112.95
± .64711
7.75
− 12.25 = 10.3000 ± 1.6240
8.00 − 12.00 = 10.3500 ±
1.49583
12.75
− 14.75 = 13.8500 ± .89443
17
Praecitrullus
fistulosus
(Stocks) Pangalo
L
575.25 − 584.00 = 578.95 ±
3.49
22.50
− 32.75 = 28.0000 ± 4.172
23.50 − 33.00 = 27.6500 ±
4.207
17.25
− 26.25 = 22.2000 ± 3.858
W
238.00 − 244.75 = 241.90 ±
2.7477
13.00
− 19.25 = 16.2500 ± 2.417
13.25 − 18.75 = 16.1000 ±
2.043
10.50
− 20.00 = 14.6000 ± 3.529
Table 4. Quantitative tendril anatomical features among Cucurbitaceous species.
Sr
No
.
Cucurbitaceous
taxa
L x W
Chlorenchyma cell (µm)
Sclerenchyma
Cell (µm)
Parenchyma
Cell (µm)
Vessel
Elements(µm)
Vascular
Bundle (µm)
1.
Citrullus colocynthis
(L) Schrad.
L
31.75 − 50.25 = 40.50±7.65
11.25 − 25 = 25±5.76
50.50 − 84.75 = 66.25±15.04
18.00 − 26.25 = 21.70±3.52
135.50 − 188 = 161.55±21.20
W
12.00 − 19.25 = 16.05±3.04
10.50 − 22.25 = 16.10±4.73
47.00 − 77.25 = 63.35±13.40
5.70 − 9.90 = 7.82±1.69
74.25 − 90.75 = 81.40±6.79
2.
Citrullus lanatus
(Thunb.)
Matsum.& Nakai
L
16.25 − 27.00 = 21±4.25
10.75 − 27.75 = 22.40±6.76
38.75 − 101 = 74.65±28.14
25.50 − 37.50 = 29.05±4.85
89.50 − 200.25 = 146.15±48.33
W
8.25
− 14.75 = 10.65±2.56
8.50
− 21 = 16.10±4.84
26.25
− 28.75 = 27.35±.96
15
− 25 = 18.30±4.44
27
− 89.75 = 64.25±29.42
3.
Cucumis melo
L.
L
18.75 − 26.25 = 22.15±2.98
16.75
− 28 = 22.10±4.83
18
− 43.75 = 29±11.15
11.25
− 16.25 = 13.75±2.26
89
− 113.75 = 99.90±10.29
W
8.75 − 11.75 = 10.35±1.23
9.75 − 22 = 14.90±5.23
9.75 − 22 = 14.90±5.23
8.50 − 11.25 = 9.55±1.10
30.50 − 55.25 = 38.20±9.97
4.
Cucumis melo subsp.
agrestis (Naudin)
Pangalo
L
15.50 − 25.25 = 20.60±4.05
8.25 − 20.25 = 13.55±4.54
20.50 − 77.75 = 53.55±26.50
9.75 − 13.25 = 11.50±1.47
47.25 − 97 = 74±19.78
W
7.75
− 10.25 = 8.9000±1.24
2.75
− 18.50 = 9±6.02
27.75
− 62.75 = 46.25±13.21
5.25
− 10.25 = 7.90±1.78
22.50
− 33.75 = 28.20±4.25
5.
L
13 − 41.25 = 25.65±10.97
5.25 − 20.50 = 16.15±6.39
23.25 − 112.50 = 61.50±37.46
7.75 − 13.75 = 11.75±2.60
75.25 − 163 = 117.10±39.43

Plants 2022, 11, 3273 8 of 29
Cucumis melo var.
flexuosus (L.)
Naudin
W
10.50
− 20 = 14.35±4.27
3.75
− 15.25 = 10.15±4.71
15.50
− 55.25 = 33.50±14.27
5.50
− 13.25 = 9.60±3.02
26.75
− 51.50 = 42.55±9.86
6.
Cucumis melo
var.
cantalupensis
Naudin
L
42.00 − 46.25 = 43.80±1.67
19.25 − 25.75 = 21.35±2.61
49.25 − 86.25 = 68.10±13.73
29.75 − 40.75 = 34.95±4.48
77.25 − 106.25 = 89.10±11.14
W
24.50
− 29.50 = 27.45±2.06
10.50
− 14.25 = 12.20±1.47
36.25
− 52.75 = 44.15±6.78
21.25
− 37.75 = 30.45±6.66
26.25
− 37.25 = 30.95±4.10
7.
Cucumis sativus
L.
L
25.50 − 62.75 = 44.25±13.87
10.25 − 30 = 19.05±8.56
13.75 − 88 = 56.95±32.34
38.25 − 47.50 = 42.55±4.09
151.75 − 301.25 = 224.15±56.12
W
13.75 − 20.25 = 17±2.54
7.75 − 20.25 = 14.25±5.60
11.00 − 39.25 = 24.45±10.77
20.50 − 38 = 29.25±7.35
101.50 − 202.25 = 125.30±43.31
8.
Cucurbita maxima
Duchesne
L
20.50 − 51.25 = 33.60±11.74
13.75 − 27.75 = 20.70±5.67
24.75 − 64.50 = 45.15±16.02
24.75 − 43 = 35.10±6.83
30.50 − 38.75 = 34±3.15
W
11.75 − 18 = 13.55±2.60
9.50 − 12.50 = 11.45±1.19
14.50 − 28.25 = 20.80±6.11
18.75 − 27.75 = 22.90±3.92
24.75 − 30.75 = 27.35±2.24
9.
Cucurbita pepo
L.
L
21.75 − 29.25 = 26.60±3.12
20.25 − 30 = 24.05±3.68
30.50 − 66.25 = 53.35±14.31
25.75 − 35.25 = 30.80±3.69
37.25 − 44.50 = 41.55±3.12
W
7.25 − 19.75 = 10.95±5.05
9.75 − 14.25 = 11.25±1.76
17.75 − 50.75 = 30.95±12.30
15.25 − 33.75 = 24.75±8.65
24.50 − 29.50 = 27.55±1.93
10.
Cucurbita pepo var.
cylindrica
L
26.75 − 46.25 = 33.55±7.57
19.50 − 25.25 = 23.05±2.34
47.25 − 98.75 = 63.45±20.67
16.25 − 22.25 = 19±2.34
22.50 − 28.75 = 26.10±2.87
W
12.50 − 28 = 19.10±7.40
12.25 − 18 = 16.35±2.32
12.25 − 25 = 18.45±5.13
11.25 − 15 = 13.30±1.52
17.25 − 21.25 = 19.35±1.51
11.
Lagenaria siceraria
(Molina) Standl.
L
20 − 46.25 = 32.50±10.08
22.50 − 31.25 = 27.60±3.43
43.75 − 105.25 = 74.15±22.10
20 − 37.50 = 28.30±7.28
63.25 − 103.75 = 86.25±17.20
W
11.75 − 17.50 = 13.40±2.38
11.75 − 20.25 = 15.05±3.65
26.75 − 36.50 = 31.40±4.32
18 − 30.75 = 24.80±5.14
25.50 − 63.50 = 38.95±14.65
12.
Luffa acutangula
(L.)Roxb.
L
17.75 − 45.50 = 30.50±10.42
15.75 − 35.75 = 26.70±8.98
24.25 − 38.75 = 31±5.76086
25.50 − 32.75 = 27.60±3.04
76.25 − 125.25 = 109.85±19.32
W
11.25 − 15.25 = 13±1.60
11.00 − 15.50 = 13.50±1.81
10 − 15.25 = 13.50±2.05
13.25 − 30 = 23.95±6.43
46.50 − 101.75 = 87.60±23.57
13.
Luffa acutangula
var. amara
C.B.Clarke
L
15.25 − 45 = 29.85±10.89
30.75 − 46 = 38.05±6.45
23.25 − 82.81 = 41.61±24.37
12.75 − 25.25 = 17±5.54
76.25 − 125.25 = 109.90±19.33
W
10.25
− 15.75 = 13.45±2.25
13.00
− 22.75 = 17.95 ±3.97
12.75
− 20.75 = 16.40±3.83±
10.25
− 22.75 = 15.80±5.58
46.50
− 101.75 = 87.60±23.57
14.
Luffa cylindrica (L)
M.Roem
L
17.50 − 37.75 = 28.80±7.97
21.25 − 31.25 = 26.50±3.99
53 − 75.75 = 65.34±9.45473
10.25 − 31.25 = 21.70±9.30
101 − 139.25 = 119.90±17.32
W
11.00 − 20.50 = 15.60±3.76
11.25 − 17.50 = 13.80±2.68
19.50 − 45.25 = 38.50±10.88
7.50 − 27.75 = 17.55±8
76 − 101.75 = 84.75±10.84
15.
Momordica charantia
L.
L
31.25 − 53.50 = 45.20±9.05
21.25 − 26.25 = 24.45±2.01
30.25 − 41.75 = 37.20±4.36
22.25 − 31 = 26.85±3.83
95.50 − 112.75 = 102.90±7.33
W
11.25 − 28.50 = 19.35±6.64
11.50 − 18 = 14.95±2.63
11.25 − 19.50 = 15.05±3.83
17.50 − 23.75 = 20.75±2.89
71 − 77.25 = 73.70±2.61
16
Momordica
balsamina L.
L
16.25 − 28.75 = 22.45±4.88
16.25 − 21.25 = 19.60±1.94
38.75 − 64 = 52.05±9.07
7.25 − 11 = 9.50±1.57
51.25 − 68.75 = 59.15±7.87
W
13.75 − 20.50 = 16.70±3.10
11.25 − 17.75 = 13.95±2.58
31.25 − 48.75 = 39.95±7.16
7.75 − 9.75 = 8.60±.741
41.25 − 50.50 = 45.90±3.90
17
Praecitrullus
fistulosus
(Stocks)
Pangalo
L
20.00 − 47.50 = 32.70±11.01
23.25 − 31.25 = 27±3.08
62.50 − 126.75 = 91.55±26
13 − 22.75 = 18±4.09
138.25 − 153.75 = 147±6.25
W
13.75
− 25.50 = 19.85±4.97
12.25
− 17.75 = 14.±2.22
20
− 75 = 41.65±27.69
10.25
− 18 = 14.35±3
104.50
− 127.75 = 118±11.78

Plants 2022, 11, 3273 9 of 29
The UPGMA clustering dendrogram for Cucurbitaceous taxa is presented in Figure
10.
Figure 10. Cluster groupings via dendrogram of Cucurbitaceous taxa based on tendril features.
Seventeen taxa of Cucurbitaceae fall into two major clusters based on the difference
in qualitative features. Similarity relationships among different Cucurbitaceous species
were explored using UPGMA clustering using tendril anatomical characters. The UPGMA
phenogram shows two main clusters, C1 and C2. The first principle cluster, C1, represents
sections C. maxima and C. pepo. The second cluster C2 further divided into two sub-clusters
comprising C2a1 of 5 species in which C. melo and C. balsamina were closely related based
on Euclidean distance mapping. The second sub-cluster, C2a2, represents ten species,
among which, based on Euclidean distance L. cylindrica and M. charantia was placed at the
minimum distance in this sub-cluster, showing the similarity in tendril qualitative fea-
tures.
Identification Keys Based on Cucurbitaceous Tendril Features
1 + Lamellar Collenchyma …………………………………………………...........................2
− Angular Collenchyma…….……………………………………………………….............5
2 + Vascular bundle with round shape, irregular tendril outline…...…….............L. siceraria
- Lamellar and angular collenchyma………………….…………………………..…...……3
3 + Irregular vascular bundle, 6-angled tendril outline………………………………..C. pepo
- Oval and irregular vascular bundle………………..………………………………..……..4
4 + C shaped tendril shape…………………………………..……….C. pepo var. cylindrica
- Angular collenchyma cell layers……..…………………………………...……………….5
5 + Tendril outline oval, vascular bundle slightly oval……..……………..……C. colocynthis
- Subsidiary type vascular bundles………………………………………………………….6
6 + V shaped tendril, elliptical shape vascular bundle…………….………………...C. lanatus
- Irregular tendril outline…………...……………………………………………………….7
7 + Subsidiary type vascular bundle, rectangular epidermal cells……………………...C. melo

Plants 2022, 11, 3273 10 of 29
- Polygonal sclerenchyma cells……………………………….……………………………..8
8 + 4-angled tendril outline, irregular vascular bundle………………..C. melo subsp. agrestis
- Irregular tendril shape…………..………………………………………………................9
9 + Tetragonal sclerenchyma cells…………………….………………C. melo var. flexuosus -
Triangular to polygonal sclerenchyma cells………..………………………………………10
10 + Central vascular bundle type..………......................................C. melo var. cantalupensis
- Elliptical and irregular vascular bundle shape…………………………………………...11
11 + Star shape tendril outline……………………………………………………….C. sativus
- Rectangular epidermal cells, irregular tendril…………………….……………………12
12 + Dumbbell type vascular bundle………………………………………………..C. maxima
- Rounded vascular bundle shape………………………..………………………………13
13 + Irregular with hollow pith tendril sape……………………..…………..…..L. acutangula
- Rounded to oval vascular bundle shape………….……………………………………14
14 + Polygonal tendril outline…………………………………...… L. acutangula var.amara
- Hexagonal tendril outline.…………………………………………15
15 + Hexagonal to polygonal chlorenchyma cells………………………….……L. cylindrica
- 4-angled tendril, rectangular to polygonal chlorenchyma cells………………………16
16 + Central type vascular bundle, tetragonal sclerenchyma cells………………M. balsamina
- Pentagonal chlorenchyma cells…………………………………………………………17
17 + Rounded and elliptical vascular bundle type……………………………M. charantia
- Rectangular epidermal cells, subsidiary vascular bundle type…………………………18
18 + Polygonal slightly U shaped tendril outline………………………………….P. fistulosus

Plants 2022, 11, 3273 11 of 29
Figure 1. (A) Field pictorial view of Citrullus colocynthis (L.) Schrad. (B) Tendril cross-section of Cit-
rullus colocynthis (L.) Schrad. (Scale bar = 10 µm) (C) Field pictorial view of Citrullus lanatus (Thunb.)
Matsum. & Nakai (D) Tendril cross section of Citrullus lanatus (Thunb.) Matsum.& Nakai (Scale bar
= 10 µm).Ad epi, Adaxial epidermis; Ab epi, Abaxial epidermis; Co, Collenchyma; Ch, Chloren-
chyma; Sc, Sclerenchyma; Pa, Parenchyma; Vb, vascular bundle.

Plants 2022, 11, 3273 12 of 29
Figure 2. (A) Field pictorial view of Cucumis melo L. (B) Tendril cross-section of Cucumis melo L.
(Scale bar = 10 µm) (C) Field pictorial view of Cucumis melo subsp. agrestis (Naudin) Pangalo (D)
Tendril cross-section of Cucumis melo subsp. agrestis (Naudin) Pangalo (Scale bar = 10 µm). Ad epi,
Adaxial epidermis; Ab epi, Abaxial epidermis; Co, Collenchyma; Ch, Chlorenchyma; Sc, Scleren-
chyma; Pa, Parenchyma; Vb, vascular bundle.

Plants 2022, 11, 3273 13 of 29
Figure 3. (A) Field pictorial view of Cucumis melo var. flexuosus (L.) Naudin (B) Tendril cross-section
of Cucumis melo var. flexuosus (L.) Naudin (Scale bar = 10 µm) (C) Field pictorial view of Cucumis
melo var.cantalupensis Naudin (D) Tendril cross-section of Cucumis melo var.cantalupensis Naudin
(Scale bar = 10 µm). Ad epi, Adaxial epidermis; Ab epi, Abaxial epidermis; Co, Collenchyma; Ch,
Chlorenchyma; Sc, Sclerenchyma; Pa, Parenchyma; Vb, vascular bundle.

Plants 2022, 11, 3273 14 of 29
Figure 4. (A) Field pictorial view of Cucumis sativus L. (B) Tendril cross-section of Cucumis sativus L.
(Scale bar = 10 µm) (C) Field pictorial view of Cucurbita maxima Duchesne (D) Tendril cross-section
of Cucurbita maxima Duchesne (Scale bar = 10 µm). Ad epi, Adaxial epidermis; Ab epi, Abaxial epi-
dermis; Co, Collenchyma; Ch, Chlorenchyma; Sc, Sclerenchyma; Pa, Parenchyma; Vb, vascular bun-
dle.

Plants 2022, 11, 3273 15 of 29
Figure 5. (A) Field pictorial view of Cucurbita pepo L. (B) Tendril cross-section of Cucurbita pepo L.
(Scale bar = 5 µm) (C) Field pictorial view of Cucurbita pepo var. cylindrica (D) Tendril cross-section
of Cucurbita pepo var. cylindrica (Scale bar = 10 µm). Ad epi, Adaxial epidermis; Ab epi, Abaxial epi-
dermis; Co, Collenchyma; Ch, Chlorenchyma; Sc, Sclerenchyma; Pa, Parenchyma; Vb, vascular bun-
dle.

Plants 2022, 11, 3273 16 of 29
Figure 6. (A) Field pictorial view of Lagenaria siceraria (Molina) Standl. (B) Tendril cross-section of
Lagenaria siceraria (Molina) Standl. (Scale bar = 5 µm) (C) Field pictorial view of Luffa acutangula (L.)
Roxb (D) Tendril cross-section of Luffa acutangula (L.) Roxb (Scale bar = 10 µm). Ad epi, Adaxial
epidermis; Ab epi, Abaxial epidermis; Co, Collenchyma; Ch, Chlorenchyma; Sc, Sclerenchyma; Pa,
Parenchyma; Vb, vascular bundle.

Plants 2022, 11, 3273 17 of 29
Figure 7. (A) Field pictorial view of Luffa acutangula var. amara C.B.Clarke (B) Tendril cross-section
of Luffa acutangula var. amara C.B.Clarke (Scale bar = 10 µm) (C) Field pictorial view of Luffa cylin-
drica(L.) M.Roem (D) Tendril cross-section of Luffa cylindrica(L.) M.Roem (Scale bar = 10 µm). Ad
epi, Adaxial epidermis; Ab epi, Abaxial epidermis; Co, Collenchyma; Ch, Chlorenchyma; Sc, Scle-
renchyma; Pa, Parenchyma; Vb, vascular bundle.

Plants 2022, 11, 3273 18 of 29
Figure 8. (A) Field pictorial view of Momordica charantia L. (B) Tendril cross-section of Momordica
charantia L. (Scale bar = 10 µm) (C) Field pictorial view of Momordica dioica Roxb. ex Willd. (D) Ten-
dril cross section of Momordica balsamina L. (Scale bar = 10 µm). Ad epi, Adaxial epidermis; Ab epi,
Abaxial epidermis; Co, Collenchyma; Ch, Chlorenchyma; Sc, Sclerenchyma; Pa, Parenchyma; Vb,
vascular bundle.

Plants 2022, 11, 3273 19 of 29
Figure 9. (A) Field pictorial view of Praecitrullus fistulosus (Stocks) Pangalo (B) Tendril cross section
of Praecitrullus fistulosus (Stocks) Pangalo (Scale bar = 10 µm). Ad epi, Adaxial epidermis; Ab epi,
Abaxial epidermis; Co, Collenchyma; Ch, Chlorenchyma; Sc, Sclerenchyma; Pa, Parenchyma; Vb,
vascular bundle.
3. Discussion
Various studies described morpho-anatomical features to classify Cucurbitaceous
species [13,14]. Different researchers have carried out the tendril anatomical investigation
of a few species of Cucurbitaceae [20] and studied the anatomical characteristics of some
species of the genus cucurbita, like flower stalk, petiole, and stem, and examined some
tendril characteristics. Leaf anatomy of Cucurbitaceous species has been briefly men-
tioned by [21], but no information is available on the tendril anatomy of Cucurbitaceous
taxa, so this study elaborates tendril histology of 12 Cucurbitaceous taxa to find out taxo-
nomic markers for their correct identification. The tendril’s shape was four and five an-
gled furrows in Momordica charantia and Cucumis sativa [22], which shows similarities with
current results. While current studies show tendrils outlined in transverse view in differ-
ent cucurbitaceous taxa are mostly irregular, slightly oval-shaped, slightly C shaped, an-
gular (four-angled, six-angled, or polygonal), and star-shaped (Table 1). The maximum
tendril length was observed in Cucurbita pepo (656.1 µm) and minimum was observed in
Momordica balsamina (123.05 µm) as shown in Figure 11. Whereas the maximum width of
tendril was noted in Cucurbita maxima (489.6 µm) and minimum in Momordica balsamina
(112.95 µm).

Plants 2022, 11, 3273 20 of 29
Figure 11. Mean tendril size variations among Cucurbitaceous taxa.
There was a single-layered epidermis present in the tendril histological section and it was predom-
inantly irregular in shape while oval-shaped cells were recorded [22]. However, [23] elaborates on
rectangular cells whereas our findings show square, oval, isodiametric, irregular, pentagonal, hex-
agonal, and polygonal types. Significant variation in the tendril micromorphology of Cucurbita-
ceous taxa was observed on both the adaxial and abaxial epidermal sides. There was variation in
the epidermal cell length and width of the studied species (Table 3). The largest cell lengthwise was
noted on the adaxial side in Praecitrullus fistulosus (28 µm) and the smallest in Luffa cylindrica (13
µm). The maximum epidermal cell width was noted in Cucumis melo var.cantalupensis (18.2 µm) and
minimum in Luffa acutangula (8.05 µm) as shown in Figure 12.
Figure 12. Graphical representation for epidermal cell size on the Adaxial surface of tendril.

Plants 2022, 11, 3273 21 of 29
Correspondingly, the largest cell length was calculated along the abaxial in Praecitrul-
lus fistulosus (27.65 µm) and the shortest in Cucumis melo subsp. agrestis (12.85 µm). The
cell width was observed to be maximum on the abaxial surface in Cucurbita pepo var. cy-
lindrica (18 µm) and minimum in Luffa acutangula var. amara (4.1 µm). Layers of scleren-
chyma and chlorenchyma (both one to eight layers) and collenchyma cells (two to six lay-
ers). However, [22] mentioned some variations in sclerenchyma two to nine, chloren-
chyma one to three and collenchyma two to six as mentioned in Figure 13.
Figure 13. Graphical representation for epidermal cell size on the abaxial surface of the tendril.
Angular collenchyma cells were observed from Iraq by [23] among Cucurbitaceae
species from Iraq, while angular lamellar types were examined in this study (Table 1). In
previous studies, two to four layers of collenchyma were present [22], while present meas-
urements revealed a distinct continuous layering of cells below the epidermis with two to
six layers of collenchyma (Table 2). The maximum layers were present in Cucurbita maxima
and Luffa acutangula var.amara both having six layers, while the minimum number of lay-
ers was present in Citrullus lanatus, with two or three layers. Collenchymatous cell size
showing maximum length in in Cucumis sativus (27.85 µm) while minimum length was in
Cucurbita pepo (12.85 µm). Whereas the largest width was calculated for Cucumis sativus
(17.7 µm) the lowest width was for Cucumis melo var. flexuosus (8.55 µm) as illustrated in
Figure 14.

Plants 2022, 11, 3273 22 of 29
Figure 14. Graphical Representation of collenchyma length and width on the surface of tendril.
There were mostly two or three layers of chlorenchyma in tendrils of Cucurbitaceae
species [22], while in recent studies, a single layer of chlorenchyma cells lies beneath col-
lenchyma but in some species, more than single layers noticed two or two to three layers
(Table 2). Maximum chlorenchymateous layers were present in Lagenaria siceraria in two
to three layers. Different shapes of chlorenchyma cells were inspected, such as rectangu-
lar, irregular, pentagonal, hexagonal, and polygonal (Table 1). The chlorenchymateous
cell size range from largest lengthwise was measured in Cucumis sativus (44.25 µm) while
the shortest was in Cucumis melo subsp. agrestis (20.6 µm). Likewise, the largest cell width-
wise was seen in Cucumis melo var. cantalupensis (27.45 µm), and the smallest cell width-
wise was observed in Cucumis melo subsp. agrestis (8.9 µm) Figure 15.
Figure 15. Graphical representation of chlorenchyma length and width on the surface of tendril.

Plants 2022, 11, 3273 23 of 29
Species like Citrullus colocynthis and Citrullus lanatus have continuous sclerenchyma
cells, similar to our findings. All the studied species illustrated the continuous scleren-
chymatous cells except Cucumis melo var catanlupensus, Cucurbita pepo var cylindrica, and
Momordica charantia, discontinuous sclerenchymatous cells were recorded in these species.
The continuous layer of sclerenchymatous cells that makes up the tendril acts as an anchor
and requires it to be robust enough to hold the weight of the plant and its fruits, especially
when it is rising [22]. The most prominent and darkly stained layers of sclernchyma cells
were just below the chlorenchyma cell layers. Sclerenchyma cell layers range from a min-
imum of two to three layers in three species of Cucumis melo subsp. agrestis, Cucumis melo
var. flexuosus, and Cucumis melo var. cantalupensis, while the maximum number of layers
was noticed in Lagenaria siceraria seven to nine layers, Table 2. Shapes of sclerenchyma
cells are dissimilar in tendrils of studied plant species. Mainly, sclerenchyma was per-
ceived as irregular, trigonal, tetragonal, hexagonal, and polygonal shaped, Table 1. There
were variations in sclerenchyma cell size ranges, maximum cell sizes lengthwise were
seen in Luffa acutangula var. amara (38.05 µm) compared to minimum cell size lengthwise
in Cucumis melo subsp. agrestis (13.55 µm). The maximum width of sclerenchyma cells was
noted in Luffa acutangula var. amara (17.95 µm), while minimum was observed in Cucumis
melo subsp. agrestis (9 µm) Figure 16.
Figure 16. Graphical representation of sclerenchyma length and width on the surface of the ten-
dril.
In earlier studies, different parenchyma cells were irregular, pentagonal, and polyg-
onal (Table 1), butt angular parenchyma cells were also recorded [23]. In the current stud-
ies, parenchymatous cells were present in all species, mostly occupying the pith region in
tendrils. The number of parenchyma cell layers differed in all studied species in Table 2.
The maximum parenchyma layers were present in Cucumis sativus six-layers, whereas the
minimum was observed in Luffa acutangula var. amara of two-layers. Parenchyma cells
were the largest cells in size present in tendrils. The largest cell lengthwise is Praecitrullus
fistulosus (91.55 µm), while mini cells were present in Cucumis melo (29 µm). The sizeable
cells, widthwise, are present in Citrullus colocynthis (63.35 µm), and compact cells width-
wise were existingin Luffa acutangula (13.5 µm) Figure 17.

Plants 2022, 11, 3273 24 of 29
Figure 17. Graphical representation of parenchyma length and width on the surface of the tendril.
Vascular bundles were mainly arranged in a subsidiary manner, and few were cen-
trally arranged in the case of Cucumis melo var. cantalupensis and Momordica charantia, Ta-
ble 1. Bicollateral-types of vascular bundles were present in tendrils. Various writers have
reported this feature in the Cucurbitaceae, which is constant across the analyzed taxa
[3,4,14,24]. Their shapes vary from oval, elliptical, rounded, irregular, and dumbbell, Ta-
ble 1. Each studied species has a different number of vascular bundles (Table 2). The max-
imum number of vascular bundles was detected in Luffa acutangula var. amara, having 12
vascular bundles, while the minimum number of vascular bundles was in Momordica bal-
samina, having three vascular bundles. Vascular bundles also vary in size. The largest vas-
cular bundle lengthwise was present in Cucumis sativus (224.25 µm), while the smallest
was observed in Cucurbita pepo var. cylindrica (26.1 µm). The largest vascular bundle
widthwise was observed in Cucumis sativus (125.3 µm), and the smallest vascular bundle
was present in Cucurbita pepo var. cylindrica (19.35 µm) Figure 18.

Plants 2022, 11, 3273 25 of 29
Figure 18. Graphical illustration showing vascular bundle size of tendril.
There was no record found about the vessel elements of the vascular bundle previ-
ously, while the current study showed a distinct number of vessel elements in the xylem
of the studied species’ tendrils. Their numbers and size vary from species to species. The
highest number of vessel elements were present in Cucumis sativus, of about 15, and the
lowest number of vessel elements existed in Cucumis melo var. cantalupensis, at around
four elements (Table 2). The biggest vessel element lengthwise was found in Cucumis sa-
tivus (42.55 µm), while the smallest vessel element lengthwise was present in Momordica
balsamina (9.5 µm). The largest vessel element widthwise was observed in Cucumis melo
var. cantalupensis (30.45 µm); meanwhile, the shortest was analyzed in Citrullus colocynthis
(7.82 µm) Figure 19.
Figure 19. Graphical variations among tendril vessel elements.

Plants 2022, 11, 3273 26 of 29
No tendril anatomical data were found about Luffa aegyptiaca. However, the species
was used against hydrocarbon-contaminated soil through rhizoremediation, and chemi-
cal analysis of this species was also carried out in research papers [25]. In Western Africa,
three genera of the Cucurbitaceae family, e.g., Momordica, Luffa, and Trichosanthes, were
studied for their foliar epidermis and tendril morphology. The significant differences in
their leaf and tendril morphology provided additional data for classifying three genera in
separate tribes [26]. Furthermore, many authors studied praecitrullus fistulosus for its me-
dicinal, anthelmintic, and anticancer activities [27–29]. Among the tendril anatomical
characters presented in the study, only a few discussed characters have been studied ear-
lier for selected species [20,22,23], while other species investigated in the current project
have not been investigated. A detailed review of the literature revealed that there is no
comprehensive study regarding the tendril anatomical features of these plants.
4. Materials and Methods
4.1. Study Site and Selected Species of Family Cucurbitaceae
This research was conducted in the desert areas of districts of Bhakkar and Layyah
in Punjab province. During March to July 2022, 17 Cucurbitaceous species each with 5
specimens were collected during field trips. Cucurbitaceous species sampling sites were
georeferenced using a GPS device (German eTrex Venture) (Table 5).
Table 5. Cucurbitaceous plant sampling along with localities, GPS coordinates, and herbarium
vouchering.
S/N Species Name Locality GPS Coordinates
Collection
Date
Voucher
Number
Accession
Number
1.
Citrullus colocynthis (L)
Schrad.
Patti
Bulanda
31°15′24.67” N
71°25′28.66” E
7-052022 QAU-NA-3 132045
2.
Citrullus lanatus (Thunb.)
Matsum.& Nakai
Mian farm
31°17′46.31” N
71°24′41.17” E 19-03-2022
QAU-NA-16
132053
3.
Cucumis melo
L. Mian farm
31°17′46.31” N
71°24′41.17” E
19-03-2022
QAU-NA-15
132059
4.
Cucumis melo subsp.
agrestis (Naudin) Pangalo
Rakhhonda
lala
31°19′01.56” N
71°25′50.80” E
07-05-2022
QAU-NA-7 132049
5.
Cucumis melo var.
flexuosus (L.) Naudin
Patti
Bulanda
31°15′24.67” N
71°25′28.66” E
08-05-2022
QAU-NA-6 132047
6.
Cucumis melo
var
.cantalupensis
Naudin
Rakhhonda
lala
31°19′01.56” N
71°25′50.80” E
07-05-2022
QAU-NA-9 132046
7.
Cucumis sativus
L. Mian farm
31°17′46.31” N
71°24′41.17” E
19-03-2022
QAU-NA-14
132060
8.
Cucurbita maxima
Duchesne
47 TDA
31°31′46.92”N
71°09′56.33”E
22-03-2022
QAU-NA-8 132052
9.
Cucurbita pepo
L. 47 TDA
31°31′46.92” N
71°09′56.33” E
20-03-2022
QAU-NA-13
132056
10.
Cucurbita pepo var.
cylindrica
Jahan khan
31°33′09.40” N
71°10′17.71” E
20-03-2022
QAU-NA-11
132051
11.
Lagenaria siceraria
(Molina) Standl.
Jahan khan
31°33′09.40” N
71°10′17.71” E 20-03-2022
QAU-NA-17
132050
12.
Luffa acutangula
(L.)Roxb
Basti Thind
31°15′31.94” N
71°27′20.64” E
22-03-2022
QAU-NA-2 132055
13.
Luffa acutangula var.amara
C.B.Clarke
Darkhana
wala
31°16′13.57” N
71°25′03.28” E
11-05-2022
QAU-NA-5 132044
14.
Luffa cylindrica(L)
M.Roem
Basti Thind
31°15′31.94” N
71°27′20.64” E
10-05-2022
QAU-NA-10
132054
15.
Momordica charantia
L. Jahan khan
31°33′09.40” N
71°10′17.71” E
20-03-2022
QAU-NA-1 132057

Plants 2022, 11, 3273 27 of 29
16.
Momordica balsamina L. 222 TDA
31°09′43.53” N
71°12′38.90” E
14-07-2022
QAU-NA-18
132143
17.
Praecitrullus fistulosus
(Stocks) Pangalo
Fateh pur
31°10′28.68” N
71°13′32.71” E
10-05-2022
QAU-NA-4 132048
The whole specimen was collected, including the tendrils, roots, stems, petioles,
leaves, and flowers. Cucurbitaceous species were grown in cultivated field crops and wild
places of the studied region. Plant specimens were pressed and dried in newspapers, iden-
tified and authenticated from the Herbarium of Pakistan (ISL). Cucurbitaceous species
names were verified from the International Plant Name Index (www.ipni.org) and Flora
of Pakistan (www.eflora.org). After preservation with ethanol and mercuric chloride so-
lution the Cucurbitaceous herbarium specimens were deposited in the ISL herbarium.
4.2. Tendril Fixation
A solution of FAA (one part of 40% formaldehyde, 18 parts of 70% ethanol, and one
part of glacial acetic acid) was used to fix mature tendrils for anatomical study for 12 h.
They were moved for 2 h in 50% ethanol and then dipped into 70% ethanol for 2 h. After-
ward, put in absolute ethanol at room temperature [22].
4.3. Histological Sectioning
Successive fixation of tendrils were sectioned using the standard technique outlined
by [30] with several changes. Tendril sections were cleaned for about a minute, and any
extra water was then removed by treating the sections with a series of alcohols ranging
from 70% to 100%. The dehydrated pieces were then submerged in xylol for one hour to
permeate the wax. At 60 °C, molten wax was used to preserve the tissues. Sections were
transferred to cast using forceps and needles, and a cast was used for this. This wax-filled
cast and its sections were chilled with cold water. The cast was afterward lifted out and
processed for microtomes. A piece of around 15–20 µm was cut from half of the length
from the base with the aid of a Shandon Microtome (Finesse 325). On a glass slide, these
sections were moved, and egg albumen was scattered all over the slide. The slide was
moved onto a hot plate and placed in an oven at 60 °C, where the wax expanded. The
tissues were extracted from the wax using xylol for five minutes. Sections were washed
thoroughly before being successively dehydrated with alcohol at concentrations of 100%,
90%, 80%, and 70%. For staining, fast green stain and Safranin O were applied. Slides were
stained for 15 to 20 min before being cleaned with distilled water. Rehydration using a
sequence of alcohol concentrations of 70%, 80%, 90%, and 100%, respectively was then
performed. To make the slide better visible, xylol was employed. DPX mountant was
placed on the slide for mounting, and the area received a cover slip. Each slide had a
proper label and was dried [31].
4.4. Tendril Micromorphology
Slides were examined using a 40x-objective LM (OPTIKA Microscope, Italy). Digital
cameras were used to capture photos of each sample under the 4, 10, and 40 objective
lenses.
4.5. Light Microscopy
A reading sheet was used to record quantitative data. 10 to 15 readings of each spe-
cies were taken under the Meiji (MT 4300H) LM at a magnification of 40×. For each species,
minimum, maximum, mean and standard deviation values of various microanatomical
parameters, including the length and width of the tendrils, the vascular bundles, collen-
chymatous cells, sclerenchyma cells, chlorenchyma cells and parenchyma cells, abaxial
and adaxial epidermal cells, and the vessel elements, were calculated [32].

Plants 2022, 11, 3273 28 of 29
4.6. Statistical Analysis
The statistical SPSS 16.0 tool was used to analyze the corresponding average data for
the measured values of tendril anatomical traits [33]. The effectiveness of the quantitative
and qualitative features were evaluated using the UPGMA clustering analysis based on
the Euclidean distance coefficient using PAST 4.03 software [34].
5. Conclusions
Through microscopic magnifications, the anatomical morphometry of tendril micro-
morphological features in Cucurbitaceous taxa exhibited variations. The resulting tendril
micromorphology provides trustworthy traits that help identify different species. Accu-
rate taxonomy will be achieved through the analysis of tendril characteristics. For taxo-
nomic examination, it is crucial to consider the shape of the tendril outline, vascular bun-
dle arrangements, sclerenchyma layers, and the morphology of the collenchyma and epi-
dermal cells. The largest collenchyma cell was found in Cucumis sativus (27.85 µm),
whereas the longest sclerenchyma cell was found in Luffa acutangula var. amara (38.05 µm).
The findings show that the taxonomic identification of these species and their relation-
ships will benefit from quantitative anatomical tendril features through clustering (UP-
GMA) analysis
Author Contributions: Conceptualization, S.M.; methodology, S.S.; validation, M.Z., M.A. and
T.M.; investigation, M.F.R.; resources, K.K.; data curation, T.B.; writing—original draft preparation,
N.A.; writing—review and editing, S.M.; visualization, Y.G. and M.K.; supervision, M.Z and M.A.;
funding, A.T.A. All authors have read and agreed to the published version of the manuscript.
Funding: Deanship of Scientific Research, Umm Al-Qura University Grant Code:
22UQU4430043DSR02.
Informed Consent Statement: Not Applicable.
Data Availability Statement: The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Acknowledgments: The authors would like to thank the Deanship of Scientific Research at Umm
Al-Qura University for supporting this work by Grant Code: 22UQU4430043DSR02.
Conflicts of Interest: The authors declare no potential conflict of interest regarding the publication
of this research work.
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