The role of explant type and selective agent application in the initial transformation rate of Lens culinaris Medik.

Lens culinaris Medik. (lentil) is an agronomically important leguminous species, but its genome modification is rarely used for obtaining new varieties, probably due to a low efficiency of transformation protocols. Development of universal, genotype-independent protocols for obtaining transgenic plants usually relies, among other factors, on the possibility of obtaining a substantial number of transgenic cells in vitro. This study aimed to adapt a previously developed Agrobacterium-mediated transformation protocol, used for a related legume, for the production of transgenic callus tissue in L. culinaris. We used two different markers of transgenic tissue, beta-glucuronidase and green fluorescent protein, to find an optimal type of explant for obtaining transgenic tissue in lentil. We also evaluated the impact of hygromycin, a common selective agent, on the amount of transgenic tissue in developing transformed explants of L. culinaris. According to our results, the transformation protocol commonly used for Medicago truncatula Gaertn. leaf explants can also be applied for obtaining transgenic calli from L. culinaris shoot apices. Explants from shoot apices demonstrated higher initial transformation rate in comparison with explants from roots, stems and leaves. Moreover, explants of different types, which were cultivated on medium without hygromycin, developed significantly fewer calli expressing reporter genes than those grown on hygromycin-containing medium, confirming that hygromycin may be used as an effective selection agent for lentil. During our analysis, we noticed GUS-like staining in calli which didn't contain plasmids for GUS gene expression. This can be explained with so-called intrinsic GUS-like activity, which was described in previous research. These data can be used for further development of effective and universal L. culinaris transformation and genome editing protocols.