Rapid Agrobacterium-mediated transformation of tobacco cotyledons using toothpicks

Tobacco is a model plant for genetic transformation, with leaf-disk transformation being the most commonly used method for its transformation. One disadvantage of leaf-disk transformation is obtaining an adequately sized leaf. Cotyledons from young seedlings are considered too small and fragile to use. In an attempt to overcome this drawback, a protocol was developed using toothpicks as a tool to inoculate cotyledons ~2mm in diameter. Agrobacterium tumefaciens LBA4404 hosting two different plasmids (pC35.BNK.2 or pRB140-Bxb1-op) was used for transformation. Fifty-six putative transgenic shoots (T0) were obtained from pC35.BNK.2 transformation. Among them, 38 (68%) grew roots in kanamycin-containing medium. Approximately, 35% of transgenic lines contained a single-copy transgenic locus based on Mendelian inheritance analysis and chi-square ({chi}2) test of T1 seedlings from 17 lines. To simplify the protocol, water-prepared Agrobacterium inoculum was used in pRB140-Bxb1-op (containing gus gene) transformation. This resulted in ~35% putative T0 transgenic lines stained strong blue with GUS histochemical staining assay. Both sets of results demonstrate toothpick inoculation to be an effective approach for Agrobacterium-mediated tobacco cotyledon transformation. This reduces wait time required in existing leaf-disk transformation method using mature leaves. Removal of step requiring submersion of explants in Agrobacterium liquid culture, the protocol also has advantages by minimizing Agrobacterium overgrowth and maintaining explant fitness for later tissue-culturing.