Nicotiana attenuata, a diploid wild tobacco, has recently became the most important model plant of Nicotiana species for studying plant-herbivore and plant-pathogen interaction, as it has relative smaller genome and shorter life cycle when compared to cultivated tobacco. However, the difficulty of rooting during tissue culture of transgenic N. attenuata seedlings greatly hampered its application as a research model in genetic engineering and molecular biology research. In order to overcome this difficulty, we tested the effects of exogenous additives, such as naphthylacetic acid (NAA), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), ancymidol, chlormequat chloride (CCC), rooting powder and active charcoal with various concentrations, on the rooting of tissue-cultured seedlings. The results revealed that the seedlings hardly produced any roots without any treatments or with CCC treatments during growth in rooting medium for 90 days, while treatments with NAA, IBA, IAA or rooting powder with appropriate concentration greatly induced rooting of tissue-cultured seedlings; Seedlings in the rooting medium supplied with 0.8 mg·L-1 IBA not only achieved a good rooting rate with above 72.73%, but also grew well in terms of morphology of roots and aboveground parts. Finally, we found that 0.8 mg·L-1 IBA treatments also greatly increased the rooting rate of transgenic seedlings, from 0% (control) to more than 60% after 20 days in rooting medium, and reached to more than 90% after wounding treatments and sub-culturing in rooting medium supplied with 0.8 mg·L-1 IBA for another 20 days. Taken all together, we successfully optimized the rooting protocol for N. attenuata transformation. The findings of this study provided a solution for the efficient induction of roots in transgenic seedlings of N. attenuata, and brought great convenience for using N. attenuata as a model plant of tobacco species.
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