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Title: Two mechanisms of lincosamide tolerance function as nuclear and plastid markers in higher plants

Massimo Bosacchi

KWS Gateway Research Center, USA

Biography

Massimo Bosacchi is a Research Scientist at the KWS Gateway Research Center.  As a member of the Cell Technologies team, he runs the maize transformation pipeline and researches cell culture-based strategies to facilitate gene editing.  He obtained his Ph.D. from Rutgers University in 2016.  Prior to this, he spent 4 years as a Research Associate with the Monsanto Company.

Abstract

Lincosamide antibiotics inhibit the regeneration of plants in tissue culture.  Resistance to lincosamides can be conferred by a mutation that modifies the ribosomal target site in the plastid genome, or by a transgene that encodes detoxification activity.  We exploited both mechanisms to recover lincosamide-tolerant events in tissue culture.  We identified several lincosamide nucleotidyltransferase (lnu) genes from medical studies and modified them for expression in plants.  Four of these genes, the lnuAN2s, lnuBs, lnuDs and lnuFs function as highly efficient selectable markers for nuclear transformation of multiple dicot species.  Under lincosamide pressure, we recovered transgenic tobacco, potato and Arabidopsis events with similar transformation frequencies as reported with kanamycin selection using the nptII gene.  In the absence of a transgene, lincosamide tolerant lines developed in tobacco tissue culture over several weeks.  Analysis of the plastid rrn23 sequence of these events revealed point mutations or 1nt insertions at the putative lincosamide binding site.  The efficiency of the lnu marker genes makes them useful additions to the toolkit of plant biologists.  Furthermore, our ability to select for a plastid trait using lincosamide antibiotics sets the stage for the development of lnu genes as plastid markers in genotypes with increased antibiotic susceptibility.