Agrobacterium-mediated transformation of tomato with the coat protein gene of tomato brown rugose fruit virus

Tomato brown rugose fruit virus (ToBRFV) has become widespread in tomato production areas in the world and Turkey in recent years. ToBRFV is easily transmitted mechanically and able to break the Tm22 resistance providing resistance to other Tobamoviruses. Since a resistance to ToBRFV has not yet been developed in commercial cultivars by conventional breeding methods, the development of alternative resistance methods should be investigated. In this study, research was conducted to develop pathogen-derived coat protein-mediated resistance against ToBRFV as an alternative resistance. For this purpose, ToBRFV-CP gene was first amplified by RT-PCR and cloned between the CaMV35S promoter and terminator to construct the gene cassette. Then, the gene cassette was cloned into the T-DNA region of the pCAMBIA0380 binary vector and transferred to Agrobacterium tumefaciens. Approximately 1970 hypocotyl and 1300 cotyledon fragments obtained from in vitro germinated tomato seeds were transformed by Agrobacterium. Approximately 350 shoots were obtained through shoot induction the transformed explants by BAP and 140 shoots survived were transferred to the rooting medium. 22 plantlets obtained from 41 of rooted shoots were transferred to glass jars containing sterile soil supplemented with liquid MS media for adaptation to the soil. Genomic DNA was isolated from nine of soil-adapted plantlets and the samples were tested by PCR method using primers specific to ToBRFV-CP gene and the gene cassette. Then, total RNA was isolated from potential transgenic plants and the expression of ToBRFV-CP transgene was analyzed by RT-qPCR methods. As a result of the expression analyses, it was determined that all tested transgenic plant expressed ToBRFV-CP gene and two transgenic plants showed high levels of expression.