Citrullus colocynthis (L.) Schrad. (colocynth): Biotechnological perspectives

Abstract

Citrullus colocynthis (L.) Schrad. is commonly known as colocynth. The fruit pulp of colocynth has medicinal properties while the seeds have nutritive qualities. C. colosynthis is resistant to high temperatures and grows in the desert regions of North Africa, the Middle East and Western Asia. C. colocynthis likely carries genes of interest that could be explored for inducing abiotic stress resistance in transgenic plants. Although the tissue culture and molecular biology of this species have been explored, the latter has been primarily used to resolve taxonomic relationships with other members of the Citrullus genus and curcubits. Genetic mining of the plant is scarce while genetic transformation protocols are also rare. The aim of the present review is to present a brief overview of the biotechnological perspectives of C. colocynthis.

Full text

Emir. J. Food Agric ● Vol 29 ● Issue 2 ● 2017 83
Citrullus colocynthis (L.) Schrad. (colocynth):
Biotechnological perspectives
Jaime A. Teixeira da Silva1*, Abdullah I. Hussain2
1P. O. Box 7, Miki-cho Post Ofce, Ikenobe 3011-2, Kagawa-ken, 761-0799, Japan, 2Department of Applied Chemistry and Biochemistry,
Natural Product and Synthetic Chemistry Laboratory, Government College University, Faisalabad 38000, Pakistan
INTRODUCTION
Citrullus colocynthis (L.) Schrad. (Cucurbitaceae) has medicinal
and ornamental purposes, the former derived primarily
from the fruit pulp (de Smet, 1997). Common names for
this plant include colocynth, bitter gourd, bitter apple, and
bitter cucumber in English while it is known as Koloquinthe
in German and coloquinte in French (de Smet, 1997).
C. colocynthis has only one accepted name but six synonyms
(The Plant List, 2017). In India and Pakistan, it is known as
tumba (Mahajan and Kumawat, 2013; Hussain et al., 2014).
Early literature indicates that C. colocynthis was the closest
relative or progenitor of watermelon (Citrullus lanatus
(South African watermelon) and Citrullus vulgaris L.
(Linnaeus’ watermelon)) (Assis et al., 2000), but molecular
phylogenetic analyses combined with herbarium sample
analyses conducted by Chomicki and Renner (2015)
revealed that in fact this was not true. Furthermore, what
was referred to as “Egusi” melon by Ntui et al. (2009, 2010a,
2010b) as Colocynthis citrullus L., may represent an incorrect
inversion of the Latin name and possibly a completely
different plant, since Chomicki and Renner (2015) indicated
that “Egusi” melon is Citrullus mucosospermus (formerly
C. lanatus subsp. mucosospermus; Levi and Thomas, 2005), a
position supported by morphological and phenetic analyses
(Achigan-Dako et al., 2015) and genetic studies (Paris, 2016)
(Table 1). Jarret and Newman (2000) also showed that
C. colocynthis and C. mucosospermus clustered separately using
internal transcribed spacer (ITS) sequences. Until formally
resolved, for all effective purposes, the authors consider,
within this review, the plant reported by Ntui et al. (2009,
2010a, 2010b) to be C. colocynthis.
Dane et al. (2007) employed three cpDNA regions and
the nuclear G3pdh transit peptide section with intron 2
in a using molecular phylogeography study to show how
C. colocynthis accessions migrated from xerophytic habitats in
Algeria, Chad and Egypt to Israel, Cyprus and the Middle
East, then further east to Iran, Afghanistan, Pakistan, and
India, whereas Moroccan accessions migrated to Australia
while Israeli accessions migrated to Ethiopia.
IMPORTANCE AND USES
According to Hussain et al. (2014), C. colocynthis has
the following traditional medicinal uses: “diabetes,
leprosy, common cold, cough, asthma, bronchitis,
Citrullus colocynthis (L.) Schrad. is commonly known as colocynth. The fruit pulp of colocynth has medicinal properties while the seeds
have nutritive qualities. C. colosynthis is resistant to high temperatures and grows in the desert regions of North Africa, the Middle East
and Western Asia. C. colocynthis likely carries genes of interest that could be explored for inducing abiotic stress resistance in transgenic
plants. Although the tissue culture and molecular biology of this species have been explored, the latter has been primarily used to resolve
taxonomic relationships with other members of the Citrullus genus and curcubits. Genetic mining of the plant is scarce while genetic
transformation protocols are also rare. The aim of the present review is to present a brief overview of the biotechnological perspectives
of C. colocynthis.
Keywords: Abiotic stress-resistance; Colocynth; Cucurbitaceae; Medicinal properties; Plant growth regulators; Tissue culture
ABSTRACT
Emirates Journal of Food and Agriculture. 2017. 29(2): 83-90
doi: 10.9755/ejfa.2016-11-1764
http://www.ejfa.me/
REVIEW ARTICLE
*Corresponding author:
Jaime A. Teixeira da Silva, P. O. Box 7, Miki-cho Post Ofce, Ikenobe 3011-2, Kagawa-ken, 761-0799, Japan. E-mail: jaimetex@yahoo.com
Received: 29 November 2016; Revised: 30 January 2017; Accepted: 31 January 2017; Published Online: 05 February 2017

Teixeira da Silva and Hussain: Citrullus colocynthis biotechnology
84 Emir. J. Food Agric ● Vol 29 ● Issue 2 ● 2017
jaundice, joint pain, cancer, toothache, wound, mastitis,
and in gastrointestinal disorders such as indigestion,
constipation, dysentery, gastroenteritis, colic pain and
different microbial infections.” Also, according to the same
authors, who wrote a comprehensive review on several
properties of C. colocynthis, indicated that there are multiple
medicinal and biological activities, including antidiabetic,
anticancer, cytotoxic, antioxidant, antilipidemic, insecticidal,
antimicrobial and anti-inammatory. De Smet (1997) also
reviewed earlier literature on the medicinal properties of
C. colocynthis.
The seeds of C. colocynthis contain edible oil, 56% of which
contained linoleic acids and 25% of which contained oleic
acids (Sawaya et al., 1983). Fruits hold bioactive chemical
constituents such as glycosides, avonoids, alkaloids and
terpenoids while “curcurbitacins A, B, C, D, E, I, J, K, and
L and Colocynthosides A, and B” have also been isolated
(Hussain et al., 2014). Several accessions have shown
resistance to several viruses and diseases (Dabauza et al.,
1997). The identication of such genes would assist in
breeding virus and disease resistance in other cucurbits
such as watermelon. To this end, Dabauza et al. (1997)
developed an Agrobacterium tumefaciens-mediated genetic
transformation protocol in which 7-day-old seedling
cotyledons were infected with strain LBA4404 carrying
the binary vector pBI121, harboring the β-glucuronidase
(gus; reporter) and the neomycin phosphotransferase
(nptII; marker) genes. Based on GUS expression, 14% of
explants were shown to be transformed. PCR conrmed
the integration of the gus and nptII genes while Southern
blot analysis conferred transmission of the gus gene to
several transgenic plants obtained by selng.
C. colocynthis is able to withstand extreme desert temperatures
through a high rate of transpiration to lower leaf
temperatures below lethal temperatures (Althawadi and
Grace, 1986). In the Thar Desert in Pakistan, the perennial
plant develops an extensive root system, and despite
only receiving only 35-40 mm of rainfall per hectare,
can produce as much as 1-1.5 t of seed, but as much as
40-fold more if rainfall is high (Mahajan and Kumawat,
2013). Exploring this ability of C. colocynthis to grow in arid
climates, Schafferman et al. (2001) assessed 28 accessions
growing wild in Negev, Arava and Sinai Deserts in Israel,
and found that the mean seed oil yield ranged from 17.1%
to 19.5% (v/w) in ve high-yielding lines (dry seed weight
basis). In addition, across the 28 accessions, linoleic acid
(C18:2) was dominant (x
- = 70.1%), followed by oleic acid
(C18:1; x
- = 13.1%), palmitic acid (C16:0; x
- = 10.1%),
and stearic acid (C18:0; x
- = 6.7%). With a seed yield of
1.5-2.1 kg/10 m2, C. colocynthis may be a potential seed oil-
yielding plant for desert and arid regions (Schafferman
et al., 2001).
C. colocynthis seeds exhibit strong dormancy, and even strong
chemical or physical treatments are unable to release the
seeds from this state of dormancy (Koller et al., 1963;
El-Hajzein and Neville, 1993), although scarication of
the testa using sandpaper resulted in 61% germination
(Saberi and Shahriari, 2011). Mahajan and Kumawat
(2013) observed 51.4% germination of seeds taken from
fresh fruits and placed at 30°C and 95% relative humidity
whereas 48 h fermentation increased germination to 58.8%,
and decreased to 16.4% after storage for 12 months at
room temperature. Sen and Bhandari (1974) achieved 98%
germination after 4.5 h of treatment with concentrated
sulphuric acid and germination in the dark since exposure
to light inhibited germination.
TISSUE CULTURE AND GENETIC
TRANSFORMATION
The success of a plant genetic transformation protocol
involves a reliable in vitro regeneration protocol (except for
in planta transformation), an effective vector to transmit
the desired gene into target tissue, stable integration and
transgene expression without transgene silencing and
only as a single gene copy (Teixeira da Silva et al., 2016).
Table 1: Taxonomy of Citrullus colocynthis (L.) Schrad*
Tanonomy and classication of Citrullus colosynthis (Renner et al. 2014; Chomicki and Renner, 2015)
Citrullus colocynthis var. capensis Alef.
Citrullus colocynthis var. insipidus Pangalo
Citrullus colocynthis subsp. insipidus (Pangalo) Fursa
Citrullus colocynthis var. lanatus (Thunb.) Matsum. & Nakai
Citrullus colocynthis var. stenotomus Pangalo
Citrullus colocynthis subsp. stenotomus (Pangalo) Fursa
English name Species
Dessert watermelon C. lanatus (Thunb.) Matsum. & Nakai
Citron watermelon C. amarus Schrad.
Egusi watermelon C. mucosospermus (Fursa) Fursa
Colocynth C. colocynthis (L.) Schrad.
*Synonyms from The Plant List (2017), and within the Citrullus genus, based on Paris (2016)

Teixeira da Silva and Hussain: Citrullus colocynthis biotechnology
Emir. J. Food Agric ● Vol 29 ● Issue 2 ● 2017 85
As described next, the rst aspect, i.e., effective in vitro
regeneration protocols, have been developed for C. colocynthis
and tissue derived from in vitro plantlets is available all-year
round and is suitable for both Agrobacterium-mediated and
bombardment-induced introduction of transgenes.
There have not been many studies published on the tissue
culture of C. colocynthis (Table 2). Dabauza et al. (1997)
induced callus and shoots from seedlings’ cotyledons, with
81.1% of explants being organogenic, and 68.3% forming
shoots, 80% of which could root on IBA-containing
medium. El-Baz et al. (2010) also used seedling tissue to
induce callus, mostly from stems, less so from leaves and
least from roots, but in all cases with more than 90% of
explants inducing callus from one tissue or another. Verma
et al. (2013) used disinfected shoot buds and nodes from
wild plants to induce shoots (as many as 18-20/explant)
and roots.
Ntui et al. (2009) induced shoots from cotyledon explants
within 12 days (4.4 shoots/explant in ‘NHC1-130’) but
hypocotyl explants failed to form shoots and only induced
callus. In ‘Ejagham’, 86.3% of explants induced shoots,
similar to improved cultivar NHC1-130. Averaged across
all four cultivars, 65% of shoots rooted on PGR-free MS
medium, and although acclimatized plants had a normal
appearance, in vitro, mixoploid and tetraploid shoots formed.
This regeneration protocol served as the basis for genetic
transformation experiments by Ntui et al. (2010b) in which
cotyledonary explants of ‘Ejagham’ and ‘NHC1-130’
were infected with Agrobacterium tumefaciens strain EHA101
harboring one of two plasmids, pIG121-Hm, carrying the
gus, hygromycin phosphotransferase (hpt) and nptII genes,
or pBBRacdS, harboring the same three genes as well as the
1-aminocyclopropane-1-carboxylate (ACC) deaminase gene.
Based on PCR of kanamycin-resistant shoots, transformation
efciency ranged from 2.4% to 9.9%, depending on the
cultivar and bacterial strain. Expanding upon this protocol,
Ntui et al. (2010a) introduced the chimeric defensin gene
from Wasabia japonica into cotyledons of ‘Ejagham’ and
‘NHC1-130’ using A. tumefaciens strain EHA101 with plasmid
pEKH1-WD, with a 25-27% transformation efciency.
Transformed plats were seen to be growing in medium in
which heavy fungal contamination was observed.
Meena et al. (2010, 2014) induced as many as 23 shoots
per shoot tip. Savitha et al. (2010a, 2010b), Shasthree et al.
(2012, 2014) and Ramakrishna and Shasthree (2016) noted
that seedling-derived leaves formed callus more than stems
(65% vs 45% of explants) while stems formed shoots
more than leaves (75% vs 51% of explants). Satyavani
et al. (2011a) induced shoots indirectly from callus, which
was induced from stem explants and a maximum of
20 shoots/explant could be induced. Taha and Mutasher
(2014) also found that seedling-derived leaves and stems
were effective (induced in 83 (leaves) - 85% (stems) of
explants) explants for callus induction. Gharehmatrossian
(2015) induced shoots indirectly from callus that had been
induced from seedling explants, but the outcome was not
quantied. More details of these studies may be found in
Table 2. Shasthree et al. (2009) found that in vitro-derived
regenerants showed considerable somaclonal variation,
including of leaves, owers, fruits and seeds.
MOLECULAR BIOLOGY AND ABIOTIC STRESS
TOLERANCE
Molecular biology in C. colocynthis research has primarily
been used in taxonomic and phylogenetic classication
through the use of molecular markers while considerable
work has been done on the characterization of genes and
transcription factors involved in abiotic stress tolerance.
Alatar et al. (2012) optimized a protocol for the isolation
of high-quality DNA from C. colocynthis from Saudi Arabia.
The extracted DNA was totally digestible with 1 U CfoI per
µg DNA and showed no detectable RNA contamination,
obtaining 10-20 µg DNA per tube in the 2-10 Kb range.
This DNA is useful for plant species fragment amplication
and microsatellite analysis.
The colocynth plant material used in experiments must
be precise identied to avoid confusion, especially form
interspecic hybrids with C. lanatus, in the eld (Fulks et al.,
1979), or as controlled crosses (Shimotsuma, 1960; Sain
et al., 2002). However, randomly amplied polymorphic
DNA (RAPD) and UPGMA cluster analyses are able
to differentiate C. colocynthis from C. lanatus (Levi et al.,
2001). RAPD was also used, alongside ISSR, to conrm
the genetic stability of in vitro regenerants (Verma et al.,
2013). Shaik et al. (2015) used two gene regions, the nuclear
G3pdh gene and the chloroplast ycf6–psbM intergenic spacer
region, to differentiate three Citrullus species (C. colocynthis,
C. lanatus (camel melon), and C. myriocarpus (prickly paddy
melon)) invasive to Australia, discovering that a western
and an eastern introduction of C. colocynthis may have taken
place. Mary et al. (2016) used DNA barcoding with the
trnH-psbA intergenic spacer to characterize C. colocynthis
relative to other curcubit genera, placing it as a distinct
phylum. Molecular markers thus serve as useful tools
in taxonomic differentiation and to assess evolutionary
events. Nimmakayala et al. (2011) offer a comprehensive
overview of the use of molecular markers in phylogeny
and taxonomy of Citrullus.
Si et al. (2009, 2010a; Table 3) found that 18 drought-
responsive genes related to abiotic and biotic stresses

Teixeira da Silva and Hussain: Citrullus colocynthis biotechnology
86 Emir. J. Food Agric ● Vol 29 ● Issue 2 ● 2017
Cultivars and
sources
Disinfection process Culture conditions* Optimal medium**,*** Reference
R309 Testa removed from seeds and
soaked in 50% commercial bleach
with 50 g/l active chlorine for 30 min.
Rinse 3 times in SDW. Cotyledons
from 7-d-old seedlings cut in half
length-wise to yield two explants
16-h PP. Grolux bulbs. 90 µmol m−2 s−1.
26±2°C. pH NR. 1% (SG) or 3% (SIM)
sucrose. 0.8% (SG) or 1% (SIM) agar.
Plant acclimatization NR
MS in darkness for 28 d (SG). MS+100 mg/L
myo-inositol+25 µM BA (CIM, SIM). MS+2.5 or 5 µM
IBA (RIM)
Dabauza et al. 1997
Three local (Nigerian)
cultivars (Ejagham,
Sewere and
Barablackedge);
one improved
cultivar (NHC1-130)
Seeds without testa dipped in
70% EtOH for 2 min, 1% NaOCl
for 20 min and rinsed with SDW.
Cotyledons of 4- or 8-d-old
seedlings cut into 1/2, 1/4 and 1/6
strips. Hypocotyls cut into 1 cm long
segments
16-h PP. 30-40 µmol m−2 s−1. 25±1°C.
pH 5.8. 1% (SG) or 3% (SIM) sucrose.
0.8% agar. Plants acclimatized in
autoclaved vermiculite
PGR-free MS (SG, RIM).
MS+5 mg/L BA (SIM).
MS+1 mg/L BA (SEM)
Ntui et al. 2009,
2010a, 2010b
Wild plants from Wadi
Soule, Sinai, Egypt
NR. Stems, leaves and roots of
2-w-old in vitro seedlings used as
explants
16-h PP. 80 µmol m−2 s−1. 24±2°C. All
other in vitro conditions NR
MS+2 or 6 mg/L 2,4-D+2 or 4 mg/L Kin; MS+0.1 mg/L
BA+5 mg/L NAA (CIM)
El-Baz et al. 2010
Mature plants growing
in Jaipur, India
Terminus of stems with shoot tips in
RTW+0.1% Labolene for 5 min then
0.05% HgCl2 for 2-3 min. Washed
with SDW 4-5×
16-h PP. LI NR. 25±2°C. pH 5.8. 3%
sucrose. 0.8% agar. Plants acclimatized
in sterilized soil : vermiculite (3:1)
MS+0.5 mg/L BA+0.5 mg/L NAA (SIM). MS+4 mg/L
IBA+0.2% AC (RIM)
Meena et al. 2010,
2014
Wild plants from
river valleys in
Andhra Pradesh, India
Leaves and stems in RTW for
5-10 min, 0.1% HgCl2 for 1-2 min,
DDW
16-h PP. 2000 lux. 25±1°C. pH 5.7.
Carbon source and gelling agent NR.
Plants acclimatized in garden soil :
FYM (1:1)
MS+1.5 or 2 mg/L 2,4-D+1 mg/L BA (or 0.5 mg/L
TDZ) (2010) or 2.5 mg/L 2,4-D+1 mg/L Zea (2014) (CIM,
SIM). MS+2 mg/L IBA+1.5 mg/L NAA (RIM)
Savitha et al. 2010a,
2010b, Shasthree
et al. 2014
Wild plants from Tamil
Nadu coast, India
Stems immersed in 70% EtOH for
1-5 min, 0.1% HgCl2 for 3 min, then
washed with SDW 4×and cut into
1.0 cm long explants
8-h PP. 20-30 µmol m−2 s−1. 25±2°C.
60±10% RH. pH 5.8. 3% sucrose. 0.8%
agar. Plants acclimatized in sterilized
soil : vermiculite (3:1)
MS+1 mg/L BA+0.5 mg/L IAA+0.5 mg/L 2,4-D (CIM).
MS+1.5 mg/L BA+0.5 mg/L NAA (SIM). MS+3 mg/L
BA+0.2% AC (RIM)
Satyavani et al.
2011a
Wild plants from
Eturnagaram Tribal,
India
Seeds germinated in vitro (protocol
NR). Leaves, stems and cotyledons
washed with 1% Labolene, RTW
for 30 min, 0.1% HgCl2 (time NR),
then washed with SDDW. Explant
size NR
pH 5.6. All other in vitro conditions NR MS+1 mg/L 2,4-D+0.5 mg/L IAA or 2 mg/L BA+0.5 mg/L
NAA (CIM, SIM)
Shasthree et al.
2012
Wild plants growing in
Jaipur, India
Shoot buds and nodal segments in
RTW for 15 min, Extran (detergent)
for 5 min then 0.1% HgCl2 for 3 min.
Washed with SDW 4-5×
16‑h PP. Cool uorescent tubes.
25 µmol m−2 s−1. 25±1°C. pH 5.8. 3%
sucrose. 0.9% agar. Plants acclimatized
in garden soil : organic manure (1:1)
MS+2.2 µM BA (SIM).
MS+4.9 µM IBA (RIM)
Verma et al. 2013
Wild plants from Iraqi
desert
Seeds in 90% EtOH for 30 s, SDW
several times, 0.5% NaOCl for
5-10 min, SDW. Leaves and stems
of 3-w old seedlings served as
explants
16-h PP. 1000 lux. 25±2°C. pH, carbon
source, gelling agent NR
PGR-free ½MS (SG). MS+2 mg/L BA+0.5 mg/L
NAA (CIM)
Taha and Mutasher
2014
Source NR. Seeds treated with 10 mg/L GA3 for
24 h. 10-d-old seedlings (all parts)
used as explants
Constant darkness (CIM) or 16-h
PP (SIM). 30°C. pH 5.7. 3% sucrose.
0.8% agar. Plants acclimatized in soil
PGR-free MS (SG). MS+1 mg/L Kin+1 mg/L IAA or
MS+2× MS vitamins+2 mg/L BA+0.1 mg/L NAA (CIM,
SIM). MS+1 mg/L IAA (RIM)
Gharehmatrossian
2015
Table 2: Tissue culture conditions for Citrullus colocynthis (L.) Schrad
(Contd...)

Teixeira da Silva and Hussain: Citrullus colocynthis biotechnology
Emir. J. Food Agric ● Vol 29 ● Issue 2 ● 2017 87
were upregulated in the shoots of seedlings of an Israeli
accession in response to PEG-induced stress. Two NAC
(no apical meristem, Arabidopsis transcription activation
factor 1 and 2, cup-shaped cotyledon 2) transcription
factors were shown to be involved in this abiotic stress
response, CcNAC1 and CcNAC2 (Wang et al., 2014a), being
inuenced by the spectral light quality (Wang et al., 2014b).
The ability to isolate and clone CcNAC1 and CcNAC2
into other plants without any drought resistance might
be an effective way of exploring arid and water-stressed
regions for expanded horticulture with drought-resistant
crops. In fact, the ability to transmit drought-resistant
signals from scion to rootstock using C. colocynthis and
watermelon (C. lanatus) (Si et al., 2010a, 2010b) holds great
promise for research on the drought-resistance mechanism
of C. colocynthis. Transcriptomic analyses of the leaves of
C. colocynthis during drought stress revealed 2545 genes
that changed signicantly during drought stress (Wang
et al., 2014c), giving promise to the mining of this plant
for drought stress-related genes.
FUTURE PERSPECTIVES
Colocynth is a rich source of functionally important
bioactive compounds and therapeutics such as polyphenols,
glycosides, triterpenes and cucurbitacins and its fruit has
been widely used for the treatment of many diseases
including diabetes, rheumatism, paronychia, ulcer and
cancer (Hussain et al., 2014). However, the biotechnology
of C. colocynthis is still underexplored. Although some tissue
culture studies and genetic transformation experiments
exist, biotechnological research into this plant would
benet from the use of the following techniques: In vitro
owering (Teixeira da Silva et al., 2014) for controlled
crosses in vitro, use of magnetic elds (Teixeira da Silva
and Dobránszki, 2015), ultrasound or sonication (Teixeira
da Silva and Dobránszki, 2014), or thin cell layers (Teixeira
da Silva and Dobránszki, 2013) to explore alternative
pathways for growth and development in vitro. Given the
heat-tolerant nature of C. colocynthis (Althawadi and Grace,
1986), and ability to grow in water stress, mining the genes
of this plant would perhaps reveal genes that could be
cloned into other plants to induce heat stress resistance.
The ability to micropropagate and mass produce uniform
plant material in vitro, independent of season, or through
the use of bioreactors, would allow callus to be constantly
harvested for silver nanoparticle production (Satyavani
et al., 2011b) with multiple uses in agriculture, medicine
and industry. The cryopreservation of C. colocynthis seeds
has already provided one such possibility for the long-term
preservation of germplasm (Alsadon et al., 2014). Using
callus that they had induced from the leaves of C. colocynthis
(Satyavani et al., 2011a), Satyavani et al. (2011b) produced
Cultivars and
sources
Disinfection process Culture conditions* Optimal medium**,*** Reference
Wild plants from
Koonoor river in
Warangal, Telangana
State, India
Similar protocol to Savitha et al.
2010a, 2010b; Shasthree et al.
2012, 2014
Similar protocol to Savitha et al. 2010a,
2010b; Shasthree et al. 2012, 2014
MS+1 mg/L 2,4-D+1.5 mg/L IBA (CIM). Callus on MS+2
mg/L NAA+1 mg/L IBA (RIM)
Ramakrishna and
Shasthree 2015
Seeds from wild
plants in Basar and
Koonoor river valleys,
Nizamabad and
Warangal, Telangana
State, India
Similar protocol to Savitha et al.
2010a, 2010b; Shasthree et al.
2012, 2014
Similar protocol to Savitha et al. 2010a,
2010b; Shasthree et al. 2012, 2014
MS+1 mg/L 2,4-D+1.5 mg/L BA (SEIM) Ramakrishna and
Shasthree 2016¶
2,4-D - 2,4-dichlorophenoxyacetic acid; AC - activated charcoal; BA - N6-benzyladenine (BA is used throughout even though BAP (6-benzylamino purine) may have been used in the original, according to Teixeira
da Silva (2012a); CIM - callus induction medium; DDW - double distilled water; EtOH - ethyl alcohol (ethanol); FYM - farmyard manure; HgCl2 - mercuric chloride; IAA - indole-3-acetic acid; IBA - indole-3-butyric
acid; Kin - kinetin (6-furfuryl aminopurine); LI - light intensity; MS - Murashige and Skoog (1962) medium; NAA - α-naphthaleneacetic acid; NaOCl - sodium hypochlorite; NR - not reported in the study; PGR - plant
growth regulator; PP - photoperiod; RH - relative humidity; RIM - root induction medium; RTW - running tap water; SDW - sterilized (by autoclaving) distilled water; SDDW - sterilized (by autoclaving) double distilled
water; SEM - shoot elongation medium; SEIM - somatic embryo induction medium; SG - seed germination; SIM - shoot induction medium; TDZ, thidiazuron (N-phenyl-N’- 1,2,3-thiadiazol-5-ylurea); w - week (s);
Zea, zeatin. *The original light intensity reported in each study has been represented since the conversion of lux to µmol m–2 s–1 is different for different illumination (main ones represented): for uorescent lamps,
1 µmol m–2 s–1=80 lux; the sun, 1 µmol m–2 s–1=55.6 lux; high voltage sodium lamp, 1 µmol m–2 s–1=71.4 lux (Thimijan and Heins 1983). **Percentage values of solids as w/v (weight/volume) and of liquids as volume/
volume (v/v). ***Even though calli was used in the original, the term callus has been used here based on the recommendation of Teixeira da Silva (2012b). ¶Claims of somatic embryogenesis without sufcient
proof (cytological, histological, genetic), i.e., only photos of macromorphology
Table 2: (Continued)

Teixeira da Silva and Hussain: Citrullus colocynthis biotechnology
88 Emir. J. Food Agric ● Vol 29 ● Issue 2 ● 2017
silver nanoparticles, which were able to reduce the toxicity
of Human epidermoid larynx carcinoma (HEp-2) cell
lines by as much as 50%. Khan et al. (2016) puried a
low molecular weight serine protease with high catalytic
activity that has many possible industrial applications from
C. colocynthis seeds.
CONCLUSIONS
Citrullus colocynthis (L.) Schrad., an important fruit species
with medicinal and nutritional value, would serve as a
valuable crop species in arid regions (Fig. 1) such as North
Africa and the Middle East. To increase production,
micropropagation protocols need to be rened and to
fortify salt- and drought-tolerance, genetic engineering
may offer a valuable solution, especially since several genes
related to drought-tolerance have already been identied.
Basic studies on the biology and biotechnology of this plant
are needed to fortify the application of molecular marker
technology, which is fairly well developed for this plant.
ACKNOWLEDGEMENTS
The authors thank Harry S. Paris (Institute of Plant
Sciences, Agricultural Research Organization, Newe Ya‘ar
Research Center, Israel), Susanne S. Renner (Department
of Biology, University of Munich, Germany) and Werner
Greuter (Herbarium Mediterraneum, Orto Botanico, Italy)
for fruitful and insightful discussion on taxonomic issues
underlying C. colocynthis and the curcubits.
Conicts of interest
The authors declare no conicts of interest.
Author contributions
Both authors contributed equally to all parts of the
development and revisions of this review.
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