An Optimized Protocol for Agrobacterium rhizogenes-Mediated Genetic Transformation of Citrullus colocynthis

Citrullus colocynthis is considered as a precious herb due to its medicinal and nutritional values and also for its ability to produce valuable bioactive compounds such as cucurbitacin E and quercetin. The hairy root systems are valuable tools for scaling-up secondary metabolites and for introducing new beneficial traits into herbs. The present research has aimed to develop a protocol for hairy root culture of C. colocynthis using Agrobacterium rhizogenes. After the establishment of the hairy root system, factors such as explant type, bacterial strain, pre-culture period, co-cultivation period, and the use of acetosyringone that affect the efficient transformation of the herb were optimized. Four A. rhizogenes strains (MSU440, A4, A13 and ATCC 15834) and three types of explant (leaf, excised shoot and hypocotyl) were tested. Furthermore, the insertion of transgene into the genome of C. colocynthis was confirmed by polymerase chain reaction analysis. The highest transformation frequency was obtained after the infection of excised shoots by MSU440. Co-cultivation for 48 hours resulted in enhanced transformation frequency, while the results of this research showed that the protocol is better not to include the pre-culturing step. In addition, the presence of acetosyringone in bacterial culture and co-cultivation medium significantly increased the success of C. colocynthis transformation. This study describes an efficient protocol for hairy roots culture of C. colocynthis which can be used for scaling-up the plant active phytochemicals or for genetic manipulations of the plant.