An efficient protocol for Agrobacterium-mediated genetic transformation of recalcitrant chrysanthemum cultivar Shinma

Chrysanthemum cultivar Shinma is a standard cultivar and has a large flower size, long vase life, and strong resistance to the white rust disease; thus, it is an important commercial cut flower in the flower markets of Korea. However, its flower color (white) is simple, so variation in flower color by using genetic transformation is necessary to increase its value in flower markets worldwide. Success of genetic transformation in chrysanthemum is dependent on many factors. In this study, factors that affect the efficient genetic transformation of this chrysanthemum were assessed, transgenic plants with the RsMYB1 anthocyanin regulatory gene were produced, and the presence of the transgene and its stable expression were confirmed using PCR and reverse transcription-PCR. Co-cultivation temperature and Agrobacterium strains were observed to be the main factors that affected higher transformation efficiency. However, the protocol developed by a combination of all optimized factors yielded eight- or fourfold higher transformation efficiency than the simple (un-optimized) protocol or individually optimized factors. The herbicide resistance assay revealed that PCR-positive transgenic shoots have stronger resistance to Basta™ than the wild type (WT). We expect that the efficient protocol developed in this study will facilitate the genetic transformation of genes of interest in this cultivar and that the anthocyanin regulatory gene will help in modifying the flower color.