Author(s)

Wei Duan¹, Liangsheng Wang², Guoqing Song^3*

¹Key Laboratory of Plant Resources/Beijing Key Laboratory of Grape Science and Enology, Institute of Botany, Chinese Academy of Sciences, Beijing, China.
²Key Laboratory of Plant Resources/Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences, Beijing, China.
³Plant Biotechnology Resource and Outreach Center, Department of Horticulture, Michigan State University, East Lansing, USA.

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 (gusA) and the neomycin phosphotransferase gene (nptII). 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 nptII and gusA 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 T₀ plants and T₁ seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii.

Regeneration, Transformation, Transgenic Plant, Nicotiana

Duan, W. , Wang, L. and Song, G. (2016) Agrobacterium tumefaciens-Mediated Transformation of Wild Tobacco Species Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa. American Journal of Plant Sciences, 7, 1-7. doi: 10.4236/ajps.2016.71001.