Agrobacterium tumefaciens -Mediated Transformation of Wild Tobacco Species Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa

Abstract

Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana deb-neyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefaciens strain EHA105 carrying the binary vector pBISN1 with an intron interrupted β-glucuronidase (GUS) reporter gene (gusA) and the neomycin phosphotransferase gene (nptII). Selection and regeneration of kanamycin resistant shoots were conducted on rege-neration medium containing 8.88 µM 6-benzylaminopurine (BAP), 0.57 µM indole-3-acetic acid (IAA), 50 mg•L −1 kanamycin and 250 mg•L −1 timentin. Kanamycin resistant shoots were rooted Murashige and Skoog (MS) medium containing 100 mg•L −1 kanamycin and 250 mg•L −1 timentin. Using this protocol, kanamycin-resistant plants were obtained from all three wild tobaccos at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa. Transcripts of nptII and gusA were detected in kanamycin-resistant T 0 transformants (i.e., 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii) by the reverse transcript polymerase chain reaction (RT-PCR), and histochemical GUS assays confirmed expression of gusA in both T 0 plants and T 1 seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii.