Genetic engineering of ORSV-resistant Phalaenopsis

Abstract

Odontoglossum ringspot virus (ORSV) is one of the most prevalent plant viruses in orchids. The aim of this research is to generate ORSV-resistant Phalaenopsis equestris var. alba overexpressing either the sense or anti-sense strand of the ORSV coat protein (CP) genes using particle bombardment. In this research, calli have been induced from different explants of P. equestris var. alba. Plant regeneration system has been established from callus. The gene comprising the capsid proteins of ORSV has been constructed, optimum conditions for particle bombardment have been investigated. Resistant calli and transgenic plantlets have been screened on selection medium. The selection medium was optimized for shoot induction with L-methionine sulfoximine (MSO) added as selection agent. Selection was done in both solid and liquid medium. It was found that liquid media and experiment showed that selection in liquid medium was faster and more efficient. PCR amplification of the inserted CP genes were used for genotyping and PCR Southern assay confirm the insertion of anti-sense strand of ORSV CP genes into several lines of P. equestris var. alba transformants. Northern blot and Western blot have also been done for the putative transformed plants, and viral inoculation and detection will performed on the transgenic Phalaenopsis when they grow to be big. Our results showed that the transgenic P. equestris var. alba has been successfully created, and hopefully, the transgenic orchids will be resistant against viral infection.