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.
Highlights
Of over 75 Nicotiana species, Nicotiana tabacum and N. benthamiana are two major model species for genetic transformation due to their high susceptibility to Agrobacterium tumefaciens and high regeneration capacity of leaf explants through shoot organogenesis [1]-[4]
Shoot Regeneration Capacity The Regeneration medium (RM) (8.88 μM BAP plus 0.57 μM indole-3-acetic acid (IAA)) used in this study is different from the commonly used one [4.44 μM BAP plus 0.54 μM α-naphthalene acetic acid (NAA)] [1] [2], it was highly efficient for shoot production from leaf explants (100% leaf explants with over 10 shoots/explants) of N. tabacum genotypes (e.g., “Xanthi” and “Samsun”) and N. benthamiana
When it was used for Nicotiana debneyi, N. clevelandii, and N. glutinosa, multiple shoots mainly from the cut edges were produced after 8 wk at frequencies of 66.7% for N. glutinosa, 95.6% for N. clevelandii, and 100% for N. debneyi
Summary
Of over 75 Nicotiana species, Nicotiana tabacum (cultivated tobacco) and N. benthamiana (wild tobacco) are two major model species for genetic transformation due to their high susceptibility to Agrobacterium tumefaciens and high regeneration capacity of leaf explants through shoot organogenesis [1]-[4]. N. tabacum (2n = 4x = 48) is an amphidiploid of interspecific hybrids and a desirable model plant for studying fundamental biological processes through functional genomics and biotechnology approaches [3]-[5]. N. clevelandii (2n = 4x = 48) is susceptible to a large number of plant viruses and N. glutinosa (2n = 2x = 24) shows resistance to a broad range of tombusviruses [11], both species are desirable wild germplasm for studying genetic control of tobmusviruses. Reliable transformation protocols for N. clevelandii, N. debneyi, and N. glutinosa, which have not been reported, will facilitate studies on disease resistance in these wild species. The protocols were applied for transformation of N. clevelandii, N. debneyi, and N. glutinosa
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