Grafting and micrografting of Nicotiana benthamiana to study the phenotypic role of nicotine

Grafting and micrografting in the model species Nicotiana benthamiana allows the study of the systemic movement of molecules and their phenotypic effects. One example involves the alkaloid nicotine, that is produced in the roots but accumulates in the leaves of this species. Mutant lines were developed in Georg Jander’s lab that lack enzymes involved in nicotine biosynthesis (the reductase A622 and Putrescine methyltransferase PMT), with the A622-deficient line (a622) showing lower nicotine levels and a cold-sensitive, dwarf phenotype leading to the hypothesis that nicotine may be related to plant growth, and therefore, that a nicotine-producing rootstock may rescue the dwarf phenotype. Grafting trials were done using wildtype, nicotine-producing and low-producing plants (a622 and pmt mutants), for which sterile seedlings growing in culture media were cut to produce scions and rootstocks that were joined in recovery plates. Later on, additions to the micrograft protocol included the use of entomological needles to fix cotyledons, scion and rootstock. A successful micrograft was obtained that was transfered to substrate and may be evaluated in the future. On the other hand, grafts were done between 4-week-old plants of the nicotine-producing and mutant lines (a622, pmt and the a622 pmt double mutant) using the transverse cut and butt alignment and the wedge-cleft methods. It is suggested to keep making grafting and micrografting trials using these and different protocols to ensure successful results that will make possible to evaluate growth of this intraspecific N. benthamiana chimeras.

I am grateful with BTI and Georg Jander for this opportunity of gaining hands-on experience in pure research with Nicotiana benthamiana, especially in evaluating grafting protocols. The feedback and weekly lab meetings within Georg’s group were essential to improve my scientific-communication skills and to know what his other students were doing. And when it comes to BTI, I thank the weekly seminars, networking events and the incredible panel of Graduate Studies that took place as unique opportunitites to know what the BTI and CALS faculty were working on. This summer was full of tremendous experiences: I felt fortunate to be surrounded by a tremendous, state-of-the-art research environment and by friends, food and fun, which will be a memory for all my life. I leave this internship with some scientific literature that I found during the development of my project, that I hope to use for my undergrad thesis in Colombia, but I also leave with an even deeper motivation to continue working on Plant-Science research.