Evaluation of virus-induced gene silencing methods for forage legumes including alfalfa, sainfoin, and fenugreek

Virus-induced gene silencing (VIGS) is a rapid reverse genetics tool that has been developed in a wide variety of plant species for assessing gene function. However, while VIGS has been utilized successfully in the diploid model leguminous species, Medicago truncatula (barrel medic), such a platform has yet to be established in forage legume crop species. Therefore, we evaluated the effectiveness of this method in forage legumes using a previously developed PEBV (pea early browning virus) system whereby a fragment of the pea (Pisum sativum L.) PHYTOENE DESATURASE (PDS) gene was transferred into a range of alfalfa (Medicago sativa L.), sainfoin (Onobrychis viciifolia Scop.) and fenugreek (Trigonella foenum-graecum L.) cultivars using leaf infiltration and apical meristem injection. Barrel medic was used as a positive control. Gene silencing was observed after 10-15 days through the presence of a leaf bleaching phenotype, and was confirmed using quantitative real-time RT-PCR. Silencing of PDS was achieved in a selection of cultivars in all species assessed, with the highest silencing efficiency apparent in fenugreek.

Over all conclusion from this study is that a highly homologous PDS gene fragment from field pea was effectively used to silence PDS genes in two perennial forage legumes (alfalfa and sainfoin) and an annual forage legume (fenugreek). The highest efficiency of silencing was achieved in fenugreek, where plasmid delivery into the host plant was achieved with more ease than the other species assessed. The VIGS-mediated introduction of a highly homologous gene fragment of the endogenous target gene represents a useful loss-of-function approach for the future characterization of potential candidate genes in forage legumes, which could increase the pace of breeding, and thus facilitate the improvement of various traits including yield and nutritional attributes in the future.