CHAPTER 1

INTRODUCTION
Introduction:
Convolvulus is derived from the Latin (convolere) meaning to entwine, and arvensis
means (of fields) (Al-Snafi,2016). It belongs to the Convolvulaceae family with 40 or more
genera and about 1,200 species. Represented in Pakistan by 13 native and cultivated genera,
C. arvensis is a major problem in wheat, common, sugarcane, potato, and other crops
(Memon & Asma,2004). Convolvulus arvensis is one of the world’s top noxious weeds and it
is found in 32 different crops in 54 countries (Holm et al.,1991).

1.1 Morphology
bindweed is a perennial vine (0.4-2 inches in height) arising from deep, persistent,
spreading roots. Leaves: are dull green with readily visible veins. Roots: Field bindweed
produces an extensive system of roots and rhizomes whitish in color, cordlike, and fleshy.
The primary root forms a taproot that can penetrate the soil to depths of two to ten feet (0.5-3
m). Lateral roots grow from buds along the taproot and form adventitious buds at the same
base (Jacobs,2007). Stems: are slender vines that run along the ground or climb any available
object. Stem length ranges from one to six feet (0.3-1.8 m), they often twine, form dense,
tangled mats, and they are normally hairless but can be pubescent. Flowers: have five fused
petals forming a 2-2.5 cm long funnel-like corolla (Al-Snafi,2016). The flowers are
hermaphrodite and pollinated by bees, flies (Kaur & Kalia,2012).

1.2 Geographical distribution

The plant is found in dry and moist soil having neutral (6.6-7.5) or alkaline pH (7.9-
8.5). The weed can survive for long drought periods due to extensive root system, but prefer
rich and fertile soils (Kaur & Kalia,2012). C. arvensis is commonly found in the salt ranges
of Pakistan and the inhabitants of the area are using this plant as folk medicine for many
generations (Iqbal et al.,2011).

1.3 Medicinal uses

The plant was reported to have used in traditional medicine system from as early as
1730s. Aerial parts of Convolvulus arvensis was used as laxative, wound healing, anti-
spasmodic anti- haemorrhagic, antiangiogenetic and for the treatment of parasites and
jaundice. In addition it was used as diuretic and in skin disorders such as anti-furunculosis,
antidandruff and in spider bites. Convolvulus arvensis was also used traditionally as
decoction in cough and flu, to treat the painful joints, inflammation and swelling (Al-
Snafi,2016). Leaves powder of Convolvulus arvensis is used to treat boils and inflammation
(Qureshi et al.,2010).
1.4 Convolvulus Classification
Scientific name: Convolvulus arvensis L.

Table 1 Classification of Convolvulus arvensis L.

 KINGDOM  PLANTAE
 PHYLUM  Magnoliophyta
 CLASS  Magnoliopsida
 ORDER  Solanales
 FAMILY  Convolvulaceae
 GENUS  Convolvulus
 SPECIES  C. arvensis L.

1.5 Need of Identification:

The issue of adulteration in medicinal plants has emerged due to the use of different
species to treat similar health problems (Shinwari et al.,2002). According to the World Health
Organization (WHO), approximately 80% of the global population relies on traditional
medicine for their primary healthcare needs (Azaizeh et al.,2003). Unfortunately, due to
similarities in the appearance of plant parts and the lack of standardized identification system,
crude medicinal plants are often tampered with or substituted during trade, leading to a loss in
effectiveness and potential toxicity (Ahmed et al.,2009).

1.6 Traditional Identification Methods:

Physical methods utilized for identification adulterants include macroscopic and
microscopic analysis. Macroscopic analysis describes plants morphology, especially for
distinguishing similar yet distinct species serving as potential adulterants. Parameters include
shape, size, color, texture, odor and surface characteristics (Choudhary&Sekon,2011).
Microscopy, observing cellular, structural, and internal tissue features, aids in discerning
between plant species (Revathy et al.,2012). It’s commonly used on powdered samples when
macroscopic features are indistinguishable. Techniques include light and advanced electron
microscopy, analyzing trichomes, starch grains, oil glands, canals, specific cell types, seeds or
pollen morphology, and vascular traces (Smilie & Khan,2010). However, microscopy based
methods have limitations in authenticated processed plant material, requiring skilled experts
and dealing with subjectivity, time consumption, challenges in distinguishing similar species,
phenological variations, expressivity, penetrance, and the lack of diagnostic morphological
features.

In recent times, molecular identification of multi-herbal products has increasingly

relied on sequencing-based techniques like DNA Barcoding. DNA technology provides a
valuable complement to chemical analysis to authenticating medicinal plants. An ideal DNA
barcode region is characterized by its short length, easy amplification, significant species
variation, high sequence efficiency, and effective species identification capability. In DNA
barcoding, universal Barcode primers (matK, rbcL, ITS, and trnH-psbA) are used to amplify
potential barcode regions, with trnH-psbA and matK emerging as the most suitable markers
for identifying plant species (Mattia et al.,2011).

1.7 DNA Barcoding: A Genomics-Based Modern Tool For

Plant Identification
Among the prevailing genome-based approaches to overcome the difficulties of
traditional taxonomy, DNA barcoding proposed by Herbert et al., (2003), is a technique has
been found to be successful in the identification of existing species and the discovery of
unknown species. It is a recent and widely used molecular and computational-based
identification system that aims to identify biological specimens and to assign them to given
species. It can be considered the core of an integrated taxonomic system (Casiraghi et al.,
2010). The DNA barcode project was initially intended as a standard method for swift and
precise animal species identification. It now covers all eukaryotic species (Herbert et
al.,2003; Miller, 2007). The emergence of DNA barcoding has improved the classification
and identification of biodiversity (Gregory, 2005). It is a technique in biodiversity research,
wherein use as a standardized region of DNA for identifying a species or a taxon. The region
used for identification is termed as a DNA barcode, which constitutes a small part (<1000bp)
of the genome and can be easily obtained. Various regions of DNA are used as markers in the
DNA barcoding process, which characterizes its universality and high resolution. For efficient
discriminatory power, a marker should necessarily show high inter- and low intra-specific
variability. This difference between inter- and intra-specific distances is known as the ‘DNA
barcoding gap’. For several years, CBOL (Consortium Barcode of Life Plant Working Group)
has focused on the identification of a universally informative plant barcode (Casiraghi et al.,
2010).

Several recommended plastid markers in plants include rpoC1, rpoB, trnH-psbA,

rbcL, matK, atpF-H, and psbK-1 (Hollingsworth et al., 2011). Of which, rbcL and matK are
suggested for DNA barcoding by the Consortium Barcode of Life Plant Working Group
(CBOL) and they are most promising coding regions in chloroplasts for plant species
identification, with low transition and transversion rates (Michel et al., 2016).

The chloroplast maturase K gene (matK) is one of the most rapidly evolving coding
regions of the plastid genome and is hypothetically the closest plant analogue to the
mitochondrial gene cytochrome oxidase 1 (CO1) used as the animal barcode (Hollingsworth
et al., 2011). Among all the plastid regions used in plants systematics, matK stands out due to
its higher rate of evolution (Barthet and Hilu, 2007; Hilu et al., 2003; Wicke and Quandt,
2009). Lahaye et al., (2008) analyzed 1084 plant species (nearly 96% of orchid species) and
showed that a portion of the plastid matK gene could be a universal DNA barcode for
flowering plants. RbcL is frequently used in phylogenetic analysis, and there are more than
50,000 sequences of it in Genbank. This gene has the benefit of being a good DNA barcoding
region at the family and genus levels, as well as being simple to amplify, sequence, and align
in most land plants (Kress et al., 2005). Molecular tools using DNA barcodes for taxonomic
identification, species delimitation, and access to phylogeny have the potential to overcome
these difficulties faced by taxonomists (Martinez-Arce et al., 2020).

1.8 Objectives
Specific objectives are as follow:
1. This study investigates DNA extraction from Leaves of Convolvulus arvensis L. by
using CTAB method.
2. To authenticate the name of Plant.
3. The main concern of this study is the correct identification of Convolvulus species.
 CHAPTER 2
Review of Literature
A number of studies have been carried out throughout the world on DNA barcoding of plants
species. A survey of review of literature was accomplished and the research work
documented in last few years is evaluated as below:

Kress et al., (2005) studied the methods for identifying species by using short
orthologous DNA sequences, known as "DNA barcodes, " have been proposed and initiated
to facilitate biodiversity studies, identifying juvenile, associate sexes, and enhance forensic
analysis. The cytochrome c oxidase 1 sequence, which has been found to be widely
applicable in animal barcoding, was not appropriate for most species of plants because of a
much slower rate of cytochrome c oxidase 1 gene evolution in higher plants than in animals.
They therefore proposed the nuclear internal transcribed spacer region and the plastid trnH-
psbA intergenic spacer as potentially usable DNA regions for applying barcoding to
flowering plants. The internal transcribed spacer is the most commonly sequenced locus used
in plant phylogenetic investigations at the species level and shows high levels of interspecific
divergence. The trnH-psbA spacer, although short (≈450-bp), is the most variable plastid
region in angiosperms and is easily amplified across a broad range of land plants. In this
paper, the comparison of the total plastid genomes of tobacco and deadly nightshade,
including closely related species in seven plant families and a group of species sampled from
a local flora encompassing 50 plant families (for a total of 99 species, 80 genera, and 53
families) were made, suggested that the sequences in this pair of loci have the potential to
discriminate among the largest number of plant species for barcoding purposes.

Hollingsworth et al., (2011) explained the process of selecting and regaining a

suitable plant barcode. They studied the factors which affect the discrimination power, some
applications of plant barcoding and some advanced projects, tools and technologies that was
required for the advancement for plant DNA barcoding. They concluded that the
discrimination success of plant barcodes is lower than that found in many animal groups such
as fishes, birds and butterflies. Despite these challenges, they elucidated some applications
such as identification of traded, ecological forensics, undertaking identifications where there
is a shortage of taxonomic expertise available, and helped in the discovery of species in some
plant’s groups. Future advancement in technology will surely lead to improvement over
current approachs, but the key step was to assemble large DNA sample steps representing the
earth's botanical diversity, supported by voucher specimens and indexed via DNA sequences.
This will provide the framework for current applications, and future developments of DNA
sequence data to kept plant species apart.

Boldt et al., (1993) examined the distribution, abundance, and economic impact of
field bindweed (Convolvulus arvensis) and hedge bindweed (Calystegia sepium) in the
continental USA. Field bindweed was found at serious densities in 957 counties, moderate in
845 counties, and low in 573 counties, across 47 states, with notable absences in Florida and
parts of the southern states. Infestations have increased in the western states since 1970 but
decreased in the Great Plains. Estimated annual crop losses due to field bindweed exceed
$377 million. Hedge bindweed was serious in 101 counties, moderate in 1109, and low in 553
across 43 states. Though less widespread, it has become more abundant since 1969 and
remains a significant issue in the midwestern and eastern USA.
Gianoli, E. (2004) studied and tested whether plants from temporally heterogeneous
habitats exhibit greater phenotypic plasticity compared to those from stable habitats. Using
Convolvulus arvensis from agrestal (oat field) and ruderal (wasteland) populations in central
Chile, it was found that plants from the agrestal habitat, which is more variable in soil
moisture, showed greater plasticity to moisture changes compared to those from the more
homogeneous ruderal habitat. Both habitats were equally variable in light intensity.
Additionally, a positive relationship between environmental stress and phenotypic integration
was observed, with this relationship being stronger in plants from the ruderal habitat. These
findings support the idea that generalist strategies are advantageous in changing
environments and highlight the importance of phenotypic integration in response to
environmental stress.

Tanveer et al., (2013) investigated how environmental factors and seed size affect
germination and seedling emergence of Convolvulus arvensis (field bindweed). Germination
occurred across a broad temperature range (15-40 ºC) with an optimum between 20-25 ºC.
High temperatures, salinity, and osmotic stress reduced germination rates and increased
germination times, although C. arvensis tolerated low levels of these stresses. Seeds placed
on the soil surface germinated best, and germination was optimal at pH 6-8, with poor
performance at highly acidic or alkaline conditions. Increased soil moisture improved
germination speed and percentage, while larger seeds germinated faster and more
successfully than smaller ones, which were more sensitive to environmental conditions.
Overall, environmental factors and seed size significantly influence germination, with larger
seeds generally providing better stand establishment and faster germination.

Austin, D. F. (2000) explained Convolvulus arvensis L., known as bindweed, is a

highly problematic weed with at least 84 common names. Native to the Mediterranean, it was
initially used medicinally along with a similar species, scammony (C. scammonia L.). While
early literature noted its medicinal use, C. arvensis spread accidentally and for ornamental
purposes. It was first recorded in North America in Virginia in the 1730s and was
commercially sold by 1807. Over time, its medicinal reputation faded, and by the 1880s, it
was recognized as a naturalized weed. Despite nearly a century of control efforts starting in
the early 1900s, effective management of this invasive species remains elusive.

Gaskin et al., (2023) studied genetic analysis of 634 Convolvulus arvensis (field
bindweed) plants from 64 populations in western North America revealed 399 unique AFLP
genotypes, with no genotypes shared between populations. Reproduction occurred via both
seeds and rhizomes, with seeds being slightly more common. Most genetic variation (54%)
was found between populations, and two genetic clusters were identified, predominating in
the western and eastern regions of the invasion. Some populations rely solely on rhizome
propagation. The study suggests that root-mining biological control agents could be effective
in managing the spread of this invasive weed.

Degennaro and Weller, (1984) researched on Convolvulus arvensis (field bindweed)

identifies five distinct biotypes within a field population, each differing in leaf morphology,
floral traits, and biomass accumulation. The biotypes also vary in their flowering times and
capacities, with some flowering much earlier and producing significantly more flowers,
indicating differences in seed production potential. The study finds that all biotypes are self-
incompatible, which affects their reproductive strategies. Additionally, there is considerable
variation in their vegetative reproduction, with differences in the percentage of root buds that
develop into shoots. This variability in growth and reproduction traits contributes to the
weed's adaptability and survival under changing environmental conditions and control
measures.

Davis et al., (2018) explained the effectiveness of various management strategies for
field bindweed (Convolvulus arvensis) and identifies research gaps. The analysis of 48
studies shows that most research focuses on short-term efficacy (within 2 years) and often
overlooks the impact on crop yield. Herbicide use is prevalent and effective in controlling
bindweed in annual cropping systems, with integrated and non-chemical methods also
proving effective. In perennial systems, biocontrol, herbicides, and non-chemical integrated
techniques work similarly well, while competition and grazing are ineffective. The study
highlights that while chemical controls are well-researched, integrated and non-chemical
methods can be equally successful. It also emphasizes the need for more long-term
monitoring to evaluate the sustainability and effectiveness of management practices.

Özkil and Üremiş, (2019) studied and investigates the germination biology of field
bindweed (Convolvulus arvensis) and three-lobe morning glory (Ipomoea triloba), focusing
on methods to break seed dormancy. Various treatments, including sulfuric acid, sodium
hydroxide, gibberellic acid, microwaving, hot water, mechanical scarification, and combined
hot+cold water, were tested. For field bindweed, sulfuric acid treatment for 90 and 120
minutes was most effective in breaking dormancy, while for three-lobe morning glory,
sulfuric acid treatment for all tested durations (15 to 120 minutes) proved effective. The study
also determined optimal germination temperatures: 10°C (minimum), 20-30°C (optimum),
and 40°C (maximum) for field bindweed; and 15°C (minimum), 25-30°C (optimum), and
40°C (maximum) for three-lobe morning glory.

Tóth and Cagáň, (2005) examined the various organisms associated with the
Convolvulaceae family worldwide and assesses their potential as biological control agents for
Convolvulus arvensis. Out of 328 associated organisms documented in 270 references, only
47 species (about 14.3%) are identified as having potential for biological control. The review
highlights specific promising candidates, including Melanagromyza albocilia (a fly),
Longitarsus pellucidus and Hypocassida subferruginea (beetles), Spermophagus sericeus (a
beetle), and the moths Emmelia trabealis and Emmelina monodactyla. The study provides
basic information on these potential biological control agents and emphasizes their relevance
for managing field bindweed.

Dall'Armellina and Zimdahl, (1988) explained how varying light levels (520, 325, and
236 μmol·m²·s⁻¹ PPFD) affect the vegetative and reproductive responses of field bindweed
and Russian knapweed. The research found that flower production for both species declined
with reduced light levels. For field bindweed, leaf area decreased with lower light, while
Russian knapweed exhibited increased leaf area at lower light intensities between 520 to 325
μmol·m²·s⁻¹ and 520 to 236 μmol·m²·s⁻¹ PPFD. The dry matter of shoots, roots, and
rhizomes in field bindweed grown from seed decreased with lower light, whereas plants
grown from rhizome segments stopped producing rhizomes entirely under low light. Russian
knapweed showed a decrease in dry matter of shoots and roots regardless of whether grown
from seed or rhizome segments. The study concludes that the total photosynthetic photon flux
density is more influential on plant responses than the sequence of light levels experienced.
 Saeed et al., (2018) studied the allelopathic potential of four wild medicinal and
aromatic plants—two Sophora species (S. mollis and S. alopecuroides), Perovskia
abrotanoides, and Peganum harmala—against field bindweed (Convolvulus arvensis). Plants
were collected from various elevations in Quetta, Balochistan, and their aqueous extracts
were tested at 4% and 32% concentrations for effects on C. arvensis germination and seedling
growth. Higher altitude plants had higher water-soluble phenolic contents and demonstrated
stronger inhibitory effects on bindweed. Among the plants, S. mollis had the highest phenolic
content, followed by P. harmala, P. abrotanoides, and S. alopecuroides. The study found that
all plant extracts significantly reduced C. arvensis germination, root, and shoot length,
suggesting these wild plants could be used as natural herbicides or as models for developing
synthetic herbicides.

Williams et al., (2014) studied a new approach called the "foundation monograph" for
incorporating DNA sequence data into large-scale taxonomic revisions, specifically focusing
on the plant genus Convolvulus. The approach aims to streamline the process of updating the
taxonomy of complex and species-rich plant groups by integrating DNA barcoding data
efficiently. In this pilot study, DNA was extracted from 310 herbarium specimens
representing 140 Convolvulus species and sequenced for three plant barcoding markers
(rbcL, matK, and ITS). The study achieved high sequencing success for these markers and
used the data to clarify species boundaries and relationships within the genus. The
phylogenetic analysis revealed that Convolvulus is paraphyletic relative to Calystegia, and
identified four main clades with strong geographical patterns. The study demonstrated that
combining rbcL, matK, and ITS barcodes provided unique identifiers for most species,
enhancing the resolution of species boundaries and aiding in the assessment of problematic
species complexes. Additionally, the study confirmed that herbarium specimens are a
valuable source of DNA for such analyses. Overall, the "foundation monograph" approach is
shown to be effective in providing a robust framework for understanding generic
relationships, character evolution, and biogeography within Convolvulus.

Kaur & Kalia (2012) studied the genus Convolvulus, part of the Convolvulaceae
family, includes many species that are problematic weeds. However, Convolvulus arvensis
has shown potential medicinal properties, such as cytotoxic activity against some tumor cell
lines. Its water extract, derived from the aerial parts, is rich in high molecular weight
proteoglycans (PGM) and exhibits strong anti-angiogenic and immune-stimulating effects.
Phytochemical analysis reveals the presence of saponins, steroids, flavonoids, alkaloids,
proteins, and lipids in this plant.

Al-Snafi (2016) explained that Convolvulus arvensis contains a variety of compounds

including alkaloids, phenolic compounds, flavonoids, carbohydrates, sugars, mucilage,
sterols, resin, tannins, unsaturated sterols/triterpenes, lactones, and proteins. In contrast,
Convolvulus scammonia is noted for its scammonin resin, dihydroxy cinnamic acid, beta-
methyl-esculetin, ipuranol, sucrose, reducing sugar, and starch. Pharmacological studies
indicate that Convolvulus arvensis has cytotoxic, antioxidant, vasorelaxant,
immunostimulant, hepatoprotective, antibacterial, antidiarrheal, and diuretic effects.
Meanwhile, Convolvulus scammonia is recognized for its purgative, vasorelaxant, anti-
platelet aggregation, anticancer, and cellular protective properties. This study will focus on
the constituents and pharmacological effects of both plants.

Gao et al., (2023) described Traditional medicine as having a rich history, but the lack
of genetic data hampered the potential of these resources. Third-generation sequencing
(TGS), like PacBio, SMRT, and Oxford Nanopore technologies, revolutionized herbal
genomics. This review highlighted TGS techniques, comparing them with Illumina, the
dominant next-gen method. It outlined nuclear and organelle genome assemblies of medicinal
plants, citing, genomics, transcriptomics, and molecular identification studies. The
advantages and drawbacks of TGS methods in medicinal contexts were discussed.
Anticipating broader use, the review envisioned TGS contributing to comprehensive genomes
and epigenomics in medicinal plants.

Schori and Showalter, (2011) explored the application of DNA barcoding for
identifying medicinal plants in Pakistan. Their research focused on developing and testing
reference DNA barcodes for a specific set of medicinal plants from the region. The study
addressed several challenges associated with the successful implementation of DNA
barcoding in plants. They collected 14 medicinal plant species, and 12 of these plants yielded
good identification sequences after DNA barcoding. The gene regions selected for the study
were rbcL, matK, and psbA-trnH. Each sequence was then submitted to GenBank's BLAST
to evaluate the similarities between the barcoding sequences of the medicinal plants and
related taxa.

Boldt et al., (1998) explained through their studies, conducted through surveys of
weed specialists and herbaria in 1994 and 1995, aimed to assess the distribution, abundance,
and economic impact of field bindweed (Convolvulus arvensis) and hedge bindweed
(Calystegia sepium) across the continental USA. It found that field bindweed is prevalent in
47 of the 48 contiguous states, with serious infestations in 957 counties, moderate in 845
counties, and low in 573 counties. Infestations have increased in western states but decreased
in the Great Plains. Crop loss due to field bindweed is estimated at over $377 million per
year. Hedge bindweed, while less widespread, is serious in 101 counties and moderate in
1109 counties, with increased abundance since 1969 and localized issues in the Midwest and
eastern states.

Schutte & Lauriault (2015) explained the potential for managing field bindweed
(Convolvulus arvensis) by using tillage to expose its roots, which can then be consumed by
ruminant livestock. It aimed to evaluate the forage nutritive value of field bindweed roots and
how their chemistry changes in response to Aceria malherbae, an eriophyid mite used for
biocontrol. Root systems from both mite-infested and non-infested plants were sampled over
three years in eastern New Mexico. The study found that Aceria malherbae reduced taproot
diameter and increased concentrations of calcium (Ca), phosphorus (P), and magnesium (Mg)
but did not affect other nutritional components such as fiber and protein. The nutritive values
of field bindweed roots were similar to those of commonly grown forages, suggesting they
could be beneficial in ruminant diets. These findings support further research on livestock
feeding on field bindweed roots and the effects of targeted root grazing on the weed.

Morin et al., (1989) studied the fungus Phomopsis convolvulus Ormeno effectively
suppressed the growth and regeneration of field bindweed in greenhouse conditions. At a
density of 108 conidia/m², it caused 95% mortality in cotyledon-stage seedlings. For 2-week-
old seedlings with three to five leaves, an inoculum density of 10 ⁹ conidia/m² resulted in 70%
mortality and significant reductions in dry weight of aboveground biomass (98%) and roots
(89%). This density also impaired the growth of 4-week-old seedlings and established plants,
though few plants were killed. In controlled studies, two inoculations were more effective
than one in reducing foliage on 4-week-old seedlings, but new shoots produced between
treatments showed less disease than expected.
 Dall'Armellina & Zimdahl (1989) investigated how watering frequency, drought
stress, and glyphosate application affect field bindweed growth. Plants grown from seed and
watered frequently had increased shoot and root dry matter, while those watered less
frequently showed no change in root runner dry matter. Conversely, plants grown from root
runner segments increased shoot and root runner dry matter with frequent watering, but root
dry matter remained unchanged. Seed-grown plants had the best regrowth with weekly
watering, while root runner segment plants regrew more with daily watering. Drought
reduced both survival rates and dry matter. Glyphosate application at 1.5 kg ai/ha effectively
prevented regrowth for 6 months, regardless of watering frequency, for plants grown for 8
weeks.

Pouresmaeil et al., (2021) studied the allelopathic effects of ethanolic extracts from
different parts of Convolvulus arvensis (root, stem, leaf, and flower) on the growth and
physiological processes of Triticum aestivum (wheat). Using a completely randomized design
with three replications, the study identified 23 compounds in the extracts, including major
ones like palmitic acid and oleic acid. The results showed that higher concentrations of the
extracts (50 and 100 g/L) reduced wheat germination and early growth, lowered
photosynthetic pigments, and increased total soluble proteins. Catalase activity increased at
low concentrations but decreased at high concentrations, while peroxidase activity decreased
overall. Malondialdehyde content, an indicator of oxidative stress, increased with higher
extract concentrations, showing oxidative damage in wheat seedlings. The study concluded
that Convolvulus arvensis extracts negatively impact wheat growth and physiological
functions, causing oxidative stress.

Mishra et al., (2016) discussed the global resurgence of herbal health systems for
maintaining well-being and highlighted the lack of standardized methods for plant species
identification in the industry. They addressed issues such as the mixing of primary ingredients
and the presence of unlabeled fillers in herbal products, which compromise product quality
and pose serious health risks to consumers. The main focus of the article was to tackle
authentication challenges in the herbal market. Despite the efforts of expert taxonomists who
tested various traditional and modern techniques, these methods proved to be time-consuming
and inefficient at the industrial level. DNA barcoding emerged as a robust tool for species
identification. Successful DNA barcodes for herbal plant identification include matK, rbcL,
trnH-psbA, ITS, trnL-F, 5S-rRNA, and 18S-rRNA, which provide sufficient information to
distinguish closely related species and identify previously ambiguous ones. DNA barcoding,
when combined with emerging technologies such as HRM analysis and multilocus strategies,
offers high-resolution species identification for the herbal drug industry. The authors
concluded that to effectively address authentication challenges in the herbal market, DNA
barcoding should be integrated with metabolomics, transcriptomics, and proteomics as
needed.
 CHAPTER 3
Materials and Method
CHAPTER 3 MATERIALS AND METHODS

3.1 Samples Collection

Convolvulus arvensis L. samples were collected from different areas of Government
Graduate College of Science Wahdat Road, Lahore. Fresh young leaves were collected from
Convolvulus for DNA extraction and Barcoding. Leaves samples were collected carefully by
using equipment to prevent contamination and any other physical damage.

3.2 Materials and chemical reagents

 Fresh young leaves
 Digital weight machine
 Beaker
 Water bath
 Distilled water
 Pestle and mortar
 Aluminum foil
 Eppendorf tubes
 Liquid nitrogen
 CTAB buffer
 B-mercaptoethanol
 Magnetic stirrer
 Measuring cylinder
 Isoamyl alcohol
 Chloroform
 Centrifuge machine
 Ethanol
 Isopropanol
 Micropipette
 TAE buffer
 Agarose gel
 Flask
 PCR machine
 Gel illuminator
 Gel plate and comb
 Ethidium bromide
 Gel electrophoresis

3.3 Practical Work

3.3.1 DNA Extraction
DNA was isolated from young Convolvulus leaves by using CTAB method. CTAB buffer was
used for DNA extraction with other chemicals. It provides high yields of best quality DNA,
especially for application like PCR, DNA sequencing, and genetic analysis. In this procedure,
various chemicals were used to isolate DNA from collected samples. There are some of the
important chemicals that were used in DNA isolation including CTAB buffer, Isoamyl
Alcohol, Chloroform, Isopropanol, Ethanol and B-mercaptoethanol. Extracted DNA was used
in PCR for amplification, DNA sequencing, Genetic analysis and Phylogenetic tree formation
for comparison and identification.

3.3.2 Preparation for DNA isolation of collected samples

1. Leaves samples were prepared to get DNA. Freshly collected leaves were washed with
distilled water to remove contaminants, dust and other impurities.
2. After washing, leaves were dried at room temperature to remove water and moisture
which can disturb DNA extraction.
3. Dried leaves samples were weighted with digital weighing machine and stored in labeling
aluminum foil to prevent any external pollutants before performing further steps.

3.3.3 Stock solution preparation

Solutions of various concentrations were prepared for DNA extraction process. Each stock
solution was labelled with its name, concentration and date of preparation. pH of buffers was
checked regularly and adjusted. Stock solutions play an essential role in the DNA extraction
process.

Table 2 Preparation of CTAB buffer for working

Chemicals Volume 50ml Volume 100ml

CTAB (10%) 10ml 20ml
Tris-chloride (2M) 2.5ml 5ml
PVP (10%) 15ml 30ml
EDTA (0.5M) 5ml 10ml
NaCl (5M) 14ml 28ml
B-mercaptoethnol 1ml 2ml
Distilled water 2.5ml 5ml

Table 3 Preparation of 50X TAE buffer

Chemicals 100ml 200ml 1000ml

Trichloride 24.2g 48.4g 242g
Acetic acid 5.71ml 11.42ml 57.1ml
EDTA (0.5) 10ml 20ml 100ml

Table 4 Preparation of 70% ethanol solution

Chemicals 25ml 50ml

Ethanol 17.5ml 35ml
Distilled water 7.5ml 15ml
 Table 5 Chloroform: Isoamyl solution preparation (24:1)

Chemicals 25ml 50ml

Chloroform 24ml 48ml
Isoamyl alcohol 1ml 2ml

Table 6 Preparation of 1X TAE buffer for use

Concentration 100ml 200ml 500ml 1000ml

50X TAE 2ml 4ml 10ml 20ml
Distilled water 98ml 196ml 490ml 980ml

3.3.4 DNA isolation process

For DNA isolation firstly, to remove any contaminants sample was washed with distilled
water and ground by using liquid nitrogen in pestle mortar. The leaves were ground
completely and converted into fine powder to proceed with the further process. Grinded
powder was shifted into Eppendorf tube and added CTAB buffer with Beta-mercaptoethanol.
Then Eppendorf tube was placed in water bath for about 45 minutes to dissolve cell walls and
lysis of other membranes.
After incubation Isoamyl alcohol and chloroform were added in the Eppendorf tube and
centrifuged it for getting aqueous layer of DNA. The aqueous layer was shifted into new
Eppendorf tube and remaining debris was discarded. Now Isopropanol was added to the
aqueous layer of DNA and placed in freezer overnight. Next step was performed as
centrifuged again and removed aqueous layer to get pellet of DNA. At the end DNA was
washed with 70% ethanol for purification. Distilled water was added to purified DNA and
mixed it completely. DNA was confirmed by loading samples in agarose gel electrophoresis
and UV light was used for visualization.

3.3.5 Agarose Gel preparation

 Gel was prepared by dissolving agarose powder in 1X TAE buffer to run DNA sample
for isolation and confirmation of DNA fragments.
 Agarose powder was mixed with buffer and heated to dissolve completely and added
ethidium bromide carefully.
 Then poured solution into gel plate in gel tray to solidify the solution.
 A gel comb was placed to create wells for sample loading.
 Agarose gel was used in electrophoresis to locate DNA fragments.
 Table 7 Preparation of agarose gel for loading DNA samples

Chemicals 25ml 50ml

Agarose powder 0.25g 0.5g
1X TAE buffer 25ml 50ml
Ethidium bromide 5µl 10µl

3.3.6 Gel electrophoresis

Gel electrophoresis is a laboratory technique used to run DNA samples by applying electric
field to move DNA samples from cathode to anode and separate DNA fragments based on
size and charge. Gel was placed in electrophoretic tray then wells were dip in 1X TAE buffer.
In the first well a leader was loaded for comparison of DNA bands. DNA sample was
prepared by mixing 5ul DNA with 3ul dye. DNA samples were loaded into the wells of
prepared agarose gel for confirmation of DNA. Electric field was applied for 30 minutes to
run DNA samples.

3.3.7 Gel illuminator

Gel illuminator is laboratory device used for the visualization of DNA and other biological
samples that are separated during the gel electrophoresis. In its UV light is used for the
confirmation of DNA samples. Gel was transferred UV transilluminator for visualization.
After transferring gel, DNA bands were observed.

3.3.8 PCR sample preparation

Table 8 PCR Sample Preparation Recipe

Chemicals Volume (µl)

dH2O 9.2µl
Taq Buffer 2.5µl
2mM dNTPs 2.5µl
10uM Forward primer 2.5µl
10uM Reverse primer 2.5µl
25mM MgCl2 3µl
Template DNA 2.5µl
Taq DNA polymerase 0.3µl
3.3.9 PCR for DNA amplification
Polymerase Chain Reaction (PCR), also known as thermal cycler is a powerful molecular
biology technique used to amplify and generate multiple copies of DNA molecules. Extracted
DNA was performed in PCR for amplification by using suitable primers such as rbcL and
matK, these two primers were found suitable for amplification of selected species DNA.
Stages of PCR as follow,

1. Pre-denaturation at 94°C for 5 minutes at one cycle

2. 40 cycles for denaturation at 94°C for 1 minute
3. Annealing at 52°C for 1 minute
4. Extension at 72°C for 1 minute
5. Final extension at 72°C at one cycle

Steps Temperature Time

Initial denaturation 72°C 5 minutes
Denaturation 94°C 1 minute
Annealing 52°C 1 minute
Extension 72°C 1 minute
Final extension 72°C 10 minutes

3.3.10 Table of primers for DNA amplification

A list of primers mentioned below that were used in DNA sample amplification process.

Table 9 primers list that are commonly used in Polymerase Chain Reaction
(PCR)
Barcode Primer Primer sequence 5’-3’ Reference
matK matK F 5’-TAATTTACGATCAATTCATTC-3’ (Ford et al.,
matK R 5’-CTTCCTCTGTAAAGAATTC-3’ 2009)
rbcL rbcL F 5’-ATGTCACCACAAACAGAAAC-3’ (Asmussen &
rbcL R 5’TCGCATGTACCYGCAGTTGC-3’ Chase, 2001)
nrITS nrITS F 5’-CCTTATCATTTAGAGGAAGGAG-3’ (Stanford et al.,
nrITS R 5’-GGAAGTAAAAGTCGTAACAAG-3’ 2000)
trnH-psbA trnH-psbA R 5’-GTTATGCATGAACGTAATGCTC-3’ (Tate &
trnH-psbA F 5’-CGCGCATGGTGGATTCACAATC-3’ Simpson,
2003)

3.3.11 Amplification of DNA with selected primers

DNA amplification was found best result under the selected primers such as rbcL and matK.
Both primers were suited for amplification of extracted DNA samples for further analysis.
 Table 10 Suitable primers for DNA amplification

Barcode Primer Primer sequence 5’-3’ Reference

matK matK F 5’-TAATTTACGATCAATTCATTC-3’ (Ford et al.,
matK R 5’-CTTCCTCTGTAAAGAATTC-3’ 2009)
rbcL rbcL F 5’-ATGTCACCACAAACAGAAAC-3’ (Asmussen &
rbcL R 5’TCGCATGTACCYGCAGTTGC-3’ Chase, 2001)

3.3.12 Suitable conditions for DNA amplification

Temperature and quality of DNA are the both most important factors.

Table 11 Selected Barcode Primer PCR Conditions

Number of Cycles Steps Temp. (°C) Time (min)

40 Initial Denaturation 94°C 3 min
40 Denaturation 94°C 40 sec
Annealing 48°C 40sec
Extension 72°C 1 min
1 Final extension 72°C 10 min
1 End Hold 4°C ∞

3.3.13 Confirmation of PCR product

The PCR product was verified by running the amplified DNA sample on a gel electrophoresis
and comparing it to a DNA ladder. The DNA ladder was placed in the first well, while the
amplified DNA was loaded into the adjacent well. An electric field was applied for
electrophoresis, and the DNA bands were then visualized under UV light.

3.3.14 Purification of PCR product

The PCR product from Convolvulus arvensis Lam. was purified for further analysis. The
purification involved phenol-chloroform extraction, followed by treatment with absolute
alcohol, chloroform, and sodium acetate, with centrifugation at each step. The DNA was then
air-dried and dissolved in ultra-pure water. At the end DNA samples were analyzed using 1%
agarose gel electrophoresis as previously described.

3.3.15 Sequencing of DNA

The amplified DNA was sent to Seoul, Korea, for DNA barcoding analysis. Next Generation
Sequencing (NGS) was used for the sequencing because of its high efficiency and accuracy.

3.3.16 Data Analysis

Data analysis was carried out using NCBI BLAST to determine genetic distances between
sequences and to perform phylogenetic analysis.

3.3.17 Genetic Distance Analysis

Analyzing differences in DNA sequences is crucial for assessing genetic variability between
species. DNA sequencing was performed using MEGA Align software to calculate this
variability. Identifying similarities between related species and their structural relationships is
essential for constructing evolutionary trees and understanding their history.

3.3.18 Phylogenetic tree Formation

DNA barcoding was employed to create phylogenetic trees for sequence alignment, which is
essential for identifying similarities between related species. Initially, DNA nucleotide
sequences were downloaded from a BLAST search, and 20 related species sequences in
FASTA format were selected. These sequences were retrieved from the NCBI-BLAST
database and imported into BioEdit software for analysis. The sequences were then aligned
using CLUSTAL-W within the EditSeq program, and the results were saved. After this, these
results were imported into MEGA format, and a Bootstrap test with 1000 replicates was
conducted to construct the phylogenetic tree using the Neighbor-Joining method.
CHAPTER 4
RESULT
CHAPTER 4 RESULT

4.1 Sample collection

Plant species samples were collected from different known areas of Punjab including Lahore,
and Govt. Graduate College of Science, Wahdat Road, Lahore where Convolvulus species are
found. Convolvulus arvensis Lam. Species was identified by its morphological taxonomic
characteristics.

Plant species samples were carefully preserved in bag and labelled with location, date, and
common name if identified plant species.

4.2 Morphological Characteristics

Table 12 General description of Convolvulus arvensis.

Botanical name Convolvulus arvensis L.

Common name Field bindweed, wild morning glory, European bindweed.
Family Convolvulaceae
Distribution in world Africa, temperate and tropical Asia and Europe.
Distribution in Pakistan All over the Pakistan
Habitat Dry gravelly field soil
Habit Weed
Life form Perennial
Flowering period Mid-summer (June to September)

4.1.2 Morphological Characters of Convolvulus arvensis L.

Stem: The stems are slender vines that run along the ground or climb any available object.
Stem length ranges from one to six feet (0.3-1.8 m), they often twine, form dense, tangled
mats, and they are normally hairless but can be pubescent.

Leaves: The leaf is ovate-oblong to lanceolate, 1.5 cm -5 cm long and 1-3 cm wide. The leaf
margin is entire and divided into three lobes, with lateral ones spreading and the middle one
ovoid elliptical, narrowly triangular, lanceolate, oblong or suborbicular. Palmate veins begin
at base of leaf and become pinnate for remainder of the leaf. The veins are depressed on the
upper surface and are raised on lower surface.

Inflorescence: inflorescence is axillary cyme composed of one or three flowers. The pedicels
are significantly longer than calyx, with hairy sepals being 2.5-5mm long. The broad funnel
shaped corolla is 15-26mm long, five lobed, white or pink, and occasionally has a pinkish and
whitish midpetaline bands. The flowers have five fused petals. Five stamens of unequal
lengths are attached to the base of corolla. The pistil is compound with thread-like stigmas.

4.3 DNA Extraction

DNA was isolated from collected plant samples by CTAB method. Samples were crushed in
the pestle and mortar in the presence of liquid nitrogen. DNA was confirmed by gel
electrophoresis process by loading DNA sample in gel and electric field was applied to run
sample in electrophoresis for 30 minutes. DNA band was observed under UV illuminator for
the confirmation of DNA in sample.

4.3 DNA Quantification Analysis

The construction of dsDNA in plants was obtained spectrophotometrically by the absorption
of 260nm wavelength using sterilized water. DNA construction was determined by using the
following formula:

DNA concentration (µg/ml) = E × OD260nm × dilution factor

(E is extinction coefficient = 50 for dsDNA)

4.5 Polymerase Chain Reaction (PCR)

The PCR process of plant DNA was performed by using DNA barcoding primers such as
rbcL, matK, nrITS, and trnH-psbA. After DNA amplification, PCR product was used in the
1% agarose gel electrophoresis. The required size band was obtained and captured with the
help of gel documentation. The rbcL primer gave positive result for selected plant.

4.6 DNA Sequences

4.6.1 Convolvulus arvensis Lam.

4.7 Genetic Sequence Distance Analysis

4.8 Phylogenetic Analysis
Phylogenetic analysis was conducted by retrieving DNA sequences of related species from a
BLAST search at NCBI. The phylogenetic tree was constructed using MEGA-11 software
with the Neighbor-Joining method, and bootstrap percentage values were employed for
analysis and validation. The analysis was based on the DNA sequence of the rbcL gene
region. The phylogenetic tree for Convolvulus arvensis Lam. species was constructed with a
maximum bootstrap value of 97%.
 CHAPTER 5
DISCUSSION
CHAPTER 5 DISCUSSION
The primary aim of this investigation is to utilize DNA barcoding for both
morphological and molecular identification of Convolvulus species. This method is
instrumental in distinguishing Convolvulus morphology across species, genus, and family
levels, while also effectively clarifying the relationships between closely and distantly related
Convolvulus samples.

The specimens for analysis were collected from various locations in Lahore at
different times. These Convolvulus samples were carefully preserved in zip-lock bags, each
labeled with the corresponding date and time of collection. Upon returning to the laboratory,
Convolvulus arvensis Lam., a member of the Convolvulaceae family, was selected for the
study. These Convolvulus species hold medicinal and nutritional significance, highlighting
the importance of precise identification. The study employed the barcode region as a tool for
accurate identification.

The DNA extraction process involved isolating genetic material from Convolvulus
species using the CTAB extraction method, followed by dissolution in distilled water. DNA
quantification was performed using both gel electrophoresis and a spectrophotometer. After
extraction, PCR was carried out on the samples using various barcode regions, including rbcL
and matK. The effectiveness of these barcode regions in distinguishing species within the
Convolvulaceae family was evaluated. Notably, the rbcL barcode region, already recognized
as a reliable primer for Convolvulus, showed superior amplification efficiency in this study,
confirming its value as an excellent barcode tool.

The amplified DNA products were sent to "CELEMICS BTseq™" in Seoul, Korea,
for sequencing using the Next Generation Sequencing method, which provides greater
accuracy than Sanger Sequencing. The resulting sequences were then analyzed using a
BLAST search on the NCBI database to identify matching sequences, followed by multiple
alignments of the amplified DNA sequences. Sequence distances were calculated using
MEGA ALIGN Software, and the existing DNA sequences in the NCBI database showed
maximum similarity with the studied sample, Convolvulus arvensis Lam.

This study aimed to analyze the distance and phylogenetic relationships among
species within the Convolvulus genus. A phylogenetic tree was constructed using MEGA-11
Software, employing the neighbor-joining method, with values below 70% omitted. The
primary objective of this research was to utilize DNA barcoding to identify Convolvulus
species collected from various locations in Lahore.
REFERENCES:

Ahmad, M., Ajabkhan, M., Zafar, M., Hasan, A., Sultana, S., Shah, G.M., & Tareen, R.B.
(2009). Chemotaxonomic authentication of herbal drug chamomile. Asian Journal of
Chemistry, 21(5), 3395.
Al-Snafi, A. E. (2016). The chemical constituents and pharmacological effects of
Convolvulus arvensis and Convolvulus scammonia-A review. IOSR Journal of
Pharmacy, 6(6), 64-75.
Austin, D. F. (2000). Bindweed (Convolvulus arvensis, Convolvulaceae) in North
America, from medicine to menace. Journal of the Torrey botanical society, 172-177.
Azaizeh, H., Fulder, S., Khalil, K., & Said, O. (2003). Ethnobotanical knowledge of local
Arab practitioners in the Middle Eastern region. Fitoterapia, 74(1-2), 98-108.
Barthet, M.M. and Hilu, K.W. (2007) Expression of matK: functional and evolutionary
implications. Am. J. Bot. 94, 1402-1412.
Boldt, P. E., Rosenthal, S. S., & Srinivasan, R. (1998). Distribution of field bindweed and
hedge bindweed in the USA. Journal of production agriculture, 11(3), 377-381.
Boldt, P. E., Rosenthal, S. S., & Srinivasan, R. (1998). Distribution of field bindweed and
hedge bindweed in the USA. Journal of production agriculture, 11(3), 377-381.
Casiraghi, M., Labra, M., Ferri, E., Galimberti, A. and Mattia, F.D. (2016) DNA
barcoding: a six-question tour to improve users' awareness about the method. Brief.
Bioinform. 11, 440-453.
Choudhary, N., & Sekhon, B.S. (2011). An overview of advances in the standardization
of herbal drugs. Journal of Pharmaceutical Education and Research, 2(2), 55.
Dall'Armellina, A. A., & Zimdahl, R. L. (1988). Effect of light on growth and
development of field bindweed (Convolvulus arvensis) and Russian knapweed
(Centaurea repens). Weed Science, 36(6), 779-783.
Dall'Armellina, A. A., & Zimdahl, R. L. (1989). Effect of watering frequency, drought,
and glyphosate on growth of field bindweed (Convolvulus arvensis). Weed science, 37(3),
314-318.
Davis, S., Mangold, J., Menalled, F., Orloff, N., Miller, Z., & Lehnhoff, E. (2018). A
meta-analysis of field bindweed (Convolvulus arvensis) management in annual and
perennial systems. Weed Science, 66(4), 540-547.
Degennaro, F. P., & Weller, S. C. (1984). Growth and reproductive characteristics of field
bindweed (Convolvulus arvensis) biotypes. Weed Science, 32(4), 525-528.
Gao, L., Xu, W., Xin, T., & Song, J. (2023). Application of third-generation sequencing to
herbal genomics. Frontiers in Plant Science, 14, 1124536.
Gaskin, J. F., Cortat, G., & West, N. M. (2023). Vegetative versus sexual reproduction
varies widely in Convolvulus arvensis across western North America. Biological
Invasions, 25(7), 2219-2229.
Gianoli, E. (2004). Plasticity of traits and correlations in two populations of Convolvulus
arvensis (Convolvulaceae) differing in environmental heterogeneity. International
Journal of Plant Sciences, 165(5), 825-832.
Gregory, T. R. (2005). DNA barcoding does not compete with taxonomy. Nature,
434(7037), 1067-1067.
Hebert, P.D.N., Cywinska, A., Ball, S.L. and deWaard, J.R. (2003a) Biological
identifications through DNA barcodes. Proc. R. Soc. Lond. B, 270, 313-321.
Herbert PDN, Cywinska A, Ball SL, deWaard JR. 2003. Biological identifications
through DNA barcodes. Proceeding of the Royal Society B: Biological Sciences 270, 313-
321.
Hilu, K.W., Borsch, T., Muller, K., Soltis, D.E., Soltis, P.S., Savolainen, V., Chase,
M.W., Powell, M.P., Alice, L.A., Evans, R., Sauquet, H., Neinhuis, C., Slotta, T.A.,
Rohwer, J.G., Campbell, C.S. and Chatrou, L.W. (2003) Angiosperm phylogeny based on
matK sequence information. Am. J. Bot. 90, 1758-1776.
Hollingsworth, P.M., Graham, S.W. and Little, D.P. (2011) Choosing and using a plant
DNA barcode. PLoS ONE, 6, e19254.
HOLM, L. G. et al. The world's worst weeds: distribution and biology. Malabar:
Krieer.The University Press of Hawaii, Honolulu, 1991. p. 98-104.
Iqbal H, Sher Z, Khan ZU. Medicinal plants from salt range Pind Dadan Khan , district
Jehlum, Punjab. Pakistan. Journal of Medicinal Plants Research, (2015); 5(11): 2157-
2168.
Jacobs, J. (2007). Ecology and management of field bindweed (Convolvulus arvensis L.).
US Department of Agriculture, Natural Resources Conservation Service.
Kaur, M., & Kalia, A. (2012). Convolvulus arvensis: A useful weed. Int J Pharm Pharm
Sci, 4(1), 38-40.
Kress, W. J., Wurdack, K. J., Zimmer, E. A., Weight,. L. A., & Janzen, D. H. (2005). Use
of DNA barcodes to identify flowering plants. Proceeding of the National Academy of
Sciences, 102(23), 8369-8374.
Lahaye, R., van der Bank, M., Bogarin, D., Warner, J., Pupulin, F., Gigot, G., Maurin, O.,
Duthoit, S., Barraclough, T.G. and Savolainen, V. (2008) DNA barcoding the floras of
biodiversity hotspots. Proc. Natl Acad. Sci. USA, 105, 2923-2928.
Martinez-Acre, A., De Jesus-Navarrete, A., and Leasi, F. (2020). DNA barcoding for
delimitation of putative Mexican marine nematodes species. Diversity 12: 107.
MEMON ASMA, R. Weed flora composition of wheat and cotton crops in District
Khairpur, Sindh. 258 f. Thesis (Ph.D. in Botânica) Shah Abdul Latif University, Pakstan,
2004.
Michel CI, Meyer RS, Taveras Y, Molina J. 2016. The nuclear internal trasncribed spacer
(ITS2) as a practical plant DNA barcode for herbal medicines. J Appl Res Med Aromat
Plants 3 (3): 94-100.
Miller SE (2007) DNA barcoding and the renaissance of taxonomy. Proceedings of the
National Academy of Sciences of the United States of America 104, 4775-4776.
Mishra, P., Kumar, A., Nagireddy, A., Mani, D.N., Shukla, A.K., Tiwari, R., &
Sundaresan, V. (2016). DNA barcoding: an efficient tool to overcome authentication
challenges in herbal market. Plant Biotechnology Journal, 14(1), 8-21.
Morin, L., Watson, A. K., & Reeleder, R. D. (1989). Efficacy of Phomopsis convolvulus
for control of field bindweed (Convolvulus arvensis). Weed Science, 37(6), 830-835.
Özkil, M., & Üremiş, İ. (2019). Research on the germination biology of field bindweed
(Convolvulus arvensis L.) and three-lobe morning glory (Ipomoea triloba L.).
Pouresmaeil, M., Motafakkerazad, R., & Sabzi Nojadeh, M. (2021). Identification of
chemical constituents and evaluation of allelopathic potential of field bindweed organs
extract on growth and physiological parameters of bread wheat. Journal of Plant
Research (Iranian Journal of Biology), 34(3), 719-732.
Qureshi R, Bhatti GR, Memon RA. Ethnomedicinal uses of herbs from the northern part
of Nara desert, Pakistan. Pak J Bot. 2010;42(2):839-51.
Revathy, S.S., Rathinamala, R., & Murugesan, M. (2012). Authentication methods for
drugs used in Ayurveda, Siddha and Unani Systems of medicine: and overview. Int J
Pharm Sci Res, 3(8), 2352-2361.
Saeed, S., Ahmed, A., Shah, S. H., Begum, S., Zeeshan, M., & Khan, W. (2018).
Phytotoxic potential of wild medicinal plants from different altitudinal gradients of
Quetta Balochistan Pakistan on Convolvulus arvensis L. Pakistan Journal of Weed
Science Research, 24(4)Schori, M., & Showalter, A.M.(2011). DNA barcoding as a means
for identifying medicinal plants of Pakistan. Pak. J. Bot, 43, 1-4.
Schutte, B. J., & Lauriault, L. (2015). Nutritive value of field bindweed (Convolvulus
arvensis) roots as a potential livestock feed and the effect of Aceria malherbae on root
components. Weed technology, 29(2), 329-334.
Shinwari, M.I. (2002). The challenges of medicinal plants of Pakistan in the New
Millennium. Hamdard Medicus, 45(2),93-100.
Similie, T.J., & Khan, I.A. (2010). A comprehensive approach to identifying and
authenticating botanical products. Clinical Pharmacology & Therapeutics, 87(2), 175186.
Tanveer, A., Tasneem, M., Khaliq, A., Javaid, M. M., & Chaudhry, M. N. (2013).
Influence of seed size and ecological factors on the germination and emergence of field
bindweed (Convolvulus arvensis). Planta daninha, 31, 39-51.
Tóth, P., & Cagáň, L. (2005). Organisms associated with the family Convolvulaceae and
their potential for biological control of Convolvulus arvensis.
Wicke, S. and Quandt, D. (2009) Universal primers for amplification of the trnK/matK
region in land plants. An. Jard. Bot. Madr. 66, 285-288.
Williams, B. R., Mitchell, T. C., Wood, J. R., Harris, D. J., Scotland, R. W., & Carine, M.
A. (2014). Integrating DNA barcode data in a monographic study of Convolvulus. Taxon,
63(6), 1287-1306.