Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY

Abstract

In vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both in vitro and ex vitro HRT were optimized in soybean using the RUBY reporter. Different parameters including A. rhizogenes strain, optical density of the bacterial cell culture (OD600), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the in vitro assay was more efficient than the ex vitro assay in terms of the percentage of induction of hairy roots and transformed roots (expressing RUBY). Nonetheless, the ex vitro technique was deemed faster and a less complicated approach. The highest transformation of RUBY was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD600 (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of RUBY through two-step ex vitro hairy root transformation. Finally, using machine learning-based modeling, optimal protocols for both assays were further defined. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.

Highly efficient Rhizobium rhizogenes-mediated hairy root transformation for gene functional study in mung bean (Vigna radiata (L.) R. Wilczek)

“…Induced hairy roots transformation through R. rhizogenes has been considered as genotype-independent approach to generate transgenic root that no significant difference in hairy root induction frequency were observed among tested cultivars (Fan et al, 2020;Cheng et al, 2021;Zhou et al, 2022). However, several reports recently indicated that genotype is a critical factor effect the transformation efficiency in R. rhizogenes mediated hairy root transformation (Aggarwal et al, 2018;Xu et al, 2020;Niazian et al, 2023). Thus, our results suggested that genotype is a critical factor should be considered to examine before establish R. rhizogenes mediated hairy root transformation in mung bean.…”

Optimization of Hairy Root Transformation and Application of RUBY as a Reporter in Lotus corniculatus

“…A visual identification technology system for gene-edited soybeans was established by utilizing RUBY as a reporter system, and transgene-free genetically edited soybean plants were successfully obtained through seed and cotyledon color [14]. Therefore, RUBY can be used as a new visual reporting system [12,15,16]. In this study, RUBY was introduced into a binary expression vector as a reporter in A. rhizogenes-mediated hairy root genetic transformation in L. corniculatus.…”

Flower color modification in Torenia fournieri by genetic engineering of betacyanin pigments

“…This system can express a single open reading frame of 2A peptide-linked betalain biosynthesis genes using a single promoter and can be used for gene expression monitoring and plant transformation. For example, RUBY was utilized for betalain engineering in various crops, including carrot [ 29 ], cotton [ 30 ], cannabis [ 31 ], soybean [ 32 ], and poplar [ 33 ]. The RUBY reporter is also used as a biotechnological tool; for example, for efficient haploid identification in maize and tomatoes [ 34 ] and genome editing in soybeans as a visual indicator [ 35 ].…”