<i>Agrobacterium tumefaciens</i>-Mediated Transformation of Wild Tobacco Species <i>Nicotiana debneyi</i>, <i>Nicotiana clevelandii</i>, and <i>Nicotiana glutinosa</i>

Abstract

Studies on Agrobacterium tumefaciens -mediated transformation of wild tobaccos Nicotiana debneyi , 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 ( gus A) and the neomycin phosphotransferase gene ( npt II). Selection and regeneration of kanamycin resistant shoots were conducted on regeneration 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 npt II and gus A were detected in kanamycin-resistant T0 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 .