TITLE:
Agrobacterium tumefaciens-Mediated Transformation of Wild Tobacco Species Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa
AUTHORS:
Wei Duan, Liangsheng Wang, Guoqing Song
KEYWORDS:
Regeneration, Transformation, Transgenic Plant, Nicotiana
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.7 No.1,
January
4,
2016
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 (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.