An Efficient Root Transformation System for Recalcitrant Vicia sativa.

Vy Nguyen,Iain R Searle

doi:10.3389/fpls.2021.781014

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.

Highlights

Common vetch (Vicia sativa) is a multi-purpose legume crop that is widely used in pastures (Sattell et al, 1998), intercropping and crop rotation regimes
5-day-old soil-grown vetch seedlings were infected with R. rhizogenes by pushing the bacterial inoculum into the hypocotyl region using a small gauge needle
We considered that the first adventitious roots 9 days after infection (DAI) might be just the result of wounding, so these roots were trimmed to promote the growth of transgenic hairy roots

Summary

Introduction

Common vetch (Vicia sativa) is a multi-purpose legume crop that is widely used in pastures (Sattell et al, 1998), intercropping and crop rotation regimes. Vetch is grown mainly in Europe, Asia, North America, and Oceania, with 54% of the production originating in Europe (FAO data, 1994–2017). Vetch seed is rich in protein, up to 32%, and has very low amounts of lipids making it an appealing food source (Mao et al, 2015). In recognition that vetch has high potential for genetic improvement, several breeding programs have been established in Europe and Australia.

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