An efficient Agrobacterium tumefaciens-mediated genetic transformation of “Egusi” melon (Colocynthis citrullus L.)

Abstract

Cotyledonary explants of two ''Egusi'' genotypes, 'Ejagham' and NHC1-130, were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harbouring genes coding for betaglucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII) or plasmid pBBRacdS harbouring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Six weeks after co-cultivation, more than 35% of explants produced shoots in both cultivars. A DNA fragment corresponding to the gus gene or the selection marker nptII was amplified from genomic DNA extracted from leaves of regenerated plant clones rooted on hormone-free MS medium containing 100 mg/l kanamycin, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of one to three copies of the gus gene. Transformation efficiencies of cultivar NHC1-130 with EHA101(pIG121-Hm) and EHA101 (pIG121-Hm, pBBRacdS) were 3.8% and 10%, respectively, which were higher than those obtained for cultivar 'Ejagham' of 2.4% and 5.7%, respectively. Co-cultivation medium containing 5 mg/l BA was effective for obtaining high transformation efficiency for both cultivars as compared with that without it.

An efficient protocol for regeneration and transformation of Symphyotrichum novi-belgii

“…A positive correlation between potential high transformation efficiency and a high regeneration capacity has been reported in e.g. safflower (Shilpa et al 2010), sunflower (Gürel and Kazan 1999) and ''Egusi'' melon (Ntui et al 2010). …”

“…The longer time for shoot appearance after co-cultivation with A281 may be attributed to the significant callus induction prior to indirect shoot formation. Generally, direct organogenesis is thought to reduce the risk of somaclonal variation and usually results in true-to type regenerants (Lee and Phillips 1988;Viterbo et al 1992;Ntui et al 2010), but the occurrence and extent of in vitro introduced genetic instability is highly genotype specific (Bennici et al 2004). …”

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

“…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.…”

“…Furthermore, what was referred to as "Egusi" melon by Ntui et al (2009Ntui et al ( , 2010aNtui et al ( , 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 (2009Ntui et al ( , 2010aNtui et al ( , 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.…”