Abstract
M. polymorpha is one of emerging models in land plants. However, only two selectable markers (hpt and aadA) for nuclear-genome transformation have been developed to date. To enrich functional genomics tools for liverwort, a novel selectable marker is required. Here, we report that the gene for M. polymorpha acetolactate synthase including mutations (MpmALS) serves as a selectable marker in liverwort. We have identified ESTs encoding ALS of 678 amino acid residues in liverwort. Compared with ALS proteins of other plant species, amino acid residues conferring the sulfonylurea herbicide (chlorsulfuron)-resistance to plants were conserved in liverwort. According to the amino acid substitutions in previous works, we have prepared MpmALS. Amino acid substitutions were introduced at three positions 207 (P to S), 198 (R to S), and 574 (W to L) in MpALS by site-directed mutagenesis. Agrobacterium-mediated transformation was conducted by using mALS as selectable marker under the control of the cauliflower mosaic virus 35S promoter, and transformants were successfully obtained on media containing 1 μM chlorsulfuron. MpmALS should be helpful to conduct functional genomics in liverwort.
