An Efficient Root Transformation System for Recalcitrant Vicia sativa

Abstract

Common vetch (Vicia sativa) is a multi-purpose legume widely used in pasture and crop rotation systems. Vetch seeds have desirable nutritional characteristics and are often used to feed ruminant animals. Although transcriptomes are available for vetch, problems with genetic transformation and plant regeneration hinder functional gene studies in this legume species. Therefore, the aim of this study was to develop a simple, efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. At first, we infected the hypocotyls of 5-day-old in vitro or in vivo, soil-grown seedlings with Rhizobium rhizogenes K599 using a stabbing method and produced transgenic hairy roots after 24 days at 19 and 50% efficiency, respectively. We later improved the hairy root transformation in vitro by infecting different explants (seedling, hypocotyl-epicotyl, and shoot) with R. rhizogenes. We observed hairy root formation at the highest efficiency in shoot and hypocotyl-epicotyl explants with 100 and 93% efficiency, respectively. In both cases, an average of four hairy roots per explant were obtained, and about 73 and 91% of hairy roots from shoot and hypocotyl-epicotyl, respectively, showed stable expression of a co-transformed marker β-glucuronidase (GUS). In summary, we developed a rapid, highly efficient, hairy root transformation method by using R. rhizogenes on vetch explants, which could facilitate functional gene analysis in common vetch.

Hairy Root Induction of Taxus baccata L. by Natural Transformation with Rhizobium rhizogenes

“…The direct inoculation ex vitro method of seedlings described in the current work was based on the research of Nguyen and Searle [52]. However, liquid culture was used as an inoculum instead of a solid culture of R. rhizogenes.…”

“…The ex vitro seedlings were inoculated using an adapted protocol from Nguyen and Searle [52]. T. baccata seedlings were placed in a 13.0 × 10.0 cm pot (Billund Potter, Billund, Denmark) containing peat, and a top 2.0 cm layer of vermiculite (Figure 2).…”

Designing Novel Strategies for Improving Old Legumes: An Overview from Common Vetch

“…Therefore, up to now the improvement in Vicia has been only reported by conventional methods. Hopeful results have only recently been achieved through the development of systems based on the use of R. rhizogenes for in vitro hairy root transformation by infecting different explants [ 161 ]. New approaches to cope with drought in legumes include the construction of transgenic legumes or Genome Editing (GE) tools, and also include the novel molecular and genomic techniques of Genome-Wide Association Studies (GWASs), Marker-Assisted Selection (MAS) with molecular markers, Genomic Selection (GS), and OMICs-based technologies by using transcriptome, genome, phenotype, proteome, and metabolome data as biotechnological tools.…”

Optimisation of root traits to provide enhanced ecosystem services in agricultural systems: A focus on cover crops

“…Alternatively, mating with a line carrying a self‐compatible locus increases successful mating between the progeny to generate homozygous lines (Dinkins et al, 2021; Riday & Krohn, 2010), but this method limits introgression of diverse genetic backgrounds. Preliminary studies in hairy vetch demonstrate that transformation may be possible but have yet to produce a stable transgenic line (Nguyen & Searle, 2022). Transformation in monocot systems has become routine in major cereal crops but is at its infancy in most cover crop varieties.…”