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

R E V I E W A R T I C L E *Corresponding author: Jaime A. Teixeira da Silva, P. O. Box 7, Miki-cho Post Office, 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-inflammatory. 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, flavonoids, 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 identification 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 confirmed the integration of the gus and nptII genes while Southern blot analysis conferred transmission of the gus gene to several transgenic plants obtained by selfing. 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 five 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 oilyielding 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 scarification 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 classification 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 first 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 efficiency 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 efficiency. 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 quantified. 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, flowers, fruits and seeds. MOLECULAR BIOLOGY AND ABIOTIC STRESS TOLERANCE Molecular biology in C. colocynthis research has primarily been used in taxonomic and phylogenetic classification 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 amplification and microsatellite analysis. The colocynth plant material used in experiments must be precise identified to avoid confusion, especially form interspecific hybrids with C. lanatus, in the field (Fulks et al., 1979), or as controlled crosses (Shimotsuma, 1960; Sain et al., 2002). However, randomly amplified 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 confirm 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 space

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-inflammatory.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, flavonoids, 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 identification 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 confirmed the integration of the gus and nptII genes while Southern blot analysis conferred transmission of the gus gene to several transgenic plants obtained by selfing.
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 five 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 m 2 , C. colocynthis may be a potential seed oilyielding 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 scarification 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).
As described next, the first 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 efficiency 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 efficiency.Transformed plats were seen to be growing in medium in which heavy fungal contamination was observed.Meena et al. (2010Meena et al. ( , 2014) ) induced as many as 23 shoots per shoot tip.Savitha et al. (2010aSavitha et al. ( , 2010b)), Shasthree et al. (2012Shasthree et al. ( , 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 quantified.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, flowers, fruits and seeds.

MOLECULAR BIOLOGY AND ABIOTIC STRESS TOLERANCE
Molecular biology in C. colocynthis research has primarily been used in taxonomic and phylogenetic classification 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 amplification and microsatellite analysis.
The colocynth plant material used in experiments must be precise identified to avoid confusion, especially form interspecific hybrids with C. lanatus, in the field (Fulks et al., 1979), or as controlled crosses (Shimotsuma, 1960;Sain et al., 2002).However, randomly amplified 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 confirm 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. (2009Si et al. ( , 2010a;; (Contd...) 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 influenced 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(Si et al., , 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 significantly 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 benefit from the use of the following techniques: In vitro flowering (Teixeira da Silva et al., 2014) for controlled crosses in vitro, use of magnetic fields (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

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 refined 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 identified.
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.) growing in a field in Faisalabad in winter (January, 2017).Despite the harsh conditions, i.e., dry, cold and arid, the plant is able to set fruit.This makes colocynth a suitable winter cash crop.All photos unpublished.

Fig 1 .
Fig 1. Colocynth (Citrullus colocynthis (L.) Schrad.)growing in a field in Faisalabad in winter (January, 2017).Despite the harsh conditions, i.e., dry, cold and arid, the plant is able to set fruit.This makes colocynth a suitable winter cash crop.All photos unpublished.

Table 3 )
found that 18 droughtresponsive genes related to abiotic and biotic stresses

Table 2 : Tissue culture conditions for Citrullus colocynthis (L.) Schrad
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 sufficient proof (cytological, histological, genetic), i.e., only photos of macromorphology

Table 2 :
Khan et al. (2016)es, 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)purified a low molecular weight serine protease with high catalytic activity that has many possible industrial applications from C. colocynthis seeds.

Table 3 : 18 drought-responsive genes coding for functional and regulatory proteins that were differentially expressed in the roots of C. colocynthis after treatment with polyethylene glycol (modified from Si et al., 2009) Name of gene sequence
Si et al. (2010b)inSi et al. (2010b), encoding a respiratory burst oxidase