Development of an in vitro hairy root induction system in different soybean cultivars for gene expression and genome editing studies

Hairy root induction system has been widely applied for studies of gene function, gene expression, and genome editing in numerous plant species. In this study, we developed and evaluated the performance of an in vitro hairy root induction system via Agrobacterium rhizogenes on several Vietnamese and international soybean cultivars. The efficacy of in vitro hairy root induction and of transformation using this system was varied and depended on soybean cultivars as well as transgenic constructs. The hairy root induction frequency of different soybean cultivars ranged from 61.67% to 100% after 5 days on culture medium. From 43.8% to 79.8% of hairy roots transformed with GFP-expressing construct showed transgene expression, while that for the construct with the gus gene was from 38.07% to 72.33%. Among tested soybean cultivars, DT26 demonstrated the highest transformation and gene expression efficacy with both investigated vectors. This hairy root induction system was further utilized for targeted knockout mutagenesis via CRISPR/Cas9 of two genes which are G03 and G09 in the DT26 soybean cultivar. Successful mutagenesis in the regions of targeted genes was confirmed by shifted and multiple bands compared to which of non-transgenic hairy root in PAGE analysis. Sequencing results of targeted regions presented various nucleotide deletions ranging between -3 bp and -25 bp in size in both two genes of interest. This study laid an important basis for future gene function studies and targeted gene editing investigations on soybean plants in Vietnam.

Technology and Application of Hairy Root Culture in Monocotyledons

Background: Hairy root culture has been widely used in the production of metabolites in dicotyledons, and a large number of food crops and medicinal plants in monocotyledons need to be developed, but there are many difficulties in the induction of hairy roots in monocotyledons. The purpose of this paper is to introduce the inducing methods, influencing factors and application of hairy roots in monocotyledons, and to promote the development of hairy root system in monocotyledons. Methods: The mechanism of action of Agrobacterium rhizogenes and the current situation of hairy root induction, induction methods and influencing factors of monocotyledons were summarized so as to provide convenience for efficient acquisition of hairy root of monocotyledons. Results: Monocotyledons are not easy to produce phenols, cells are prone to lignification, adverse differentiation and selective response to Agrobacterium rhizogenes strains. It is proposed that before induction, plant varieties and explants should be selected, and different infection strains should be screened. In the process of hairy root induction, exogenous inducers such as acetosyringone can be added. Although these factors can provide some help for the induction of hairy roots in monocotyledons, we still need to pay attention to the disadvantages of monocotyledons from dicotyledons at the cellular level. Conclusion: A large number of food crops and medicinal plants are monocotyledons. Hairy root culture can be used to help the breeding and production of medicinal substances. Therefore, it is necessary to pay attention to the selection of varieties and explants, the selection of Agrobacterium rhizogenes and the addition of acetosyringone in the process of hairy root induction so as to improve the production efficiency and facilitate the development and utilization of monocotyledons.

Evaluation of influence of different strains of Agrobacterium rhizogenes on efficiency of hairy root induction in Rauwolfia serpentina

An improved protocol was established for hairy root induction in Rauwolfia serpentina using two strains of Agrobacterium rhizogenes MTCC 532 and 2364. Hairy root induction efficiency was standardized based on infection time, co-cultivation period, OD600 and acetosyringone concentration. Higher transformation efficiency was established using MTCC 532 (31%) and MTCC 2364 (24%) with 30 min infection time, 72 h co-cultivation period and 0.6 OD600. Transformation efficiency was further enhanced to 55 % with 125 μM acetosyringone. MTCC 532 was proven a better strain over the MTCC 2364 in all the tested factors. Putative transgenic hairy roots were confirmed by polymerase chain reaction using rolA specific primers. Biomass of randomly selected 5 hairy root lines was also significantly enhanced. No significant difference of growth was recorded among the lines except line no 4. In the present study, an enhanced system for Agrobacterium rhizogenesmediated hairy root culture was established which offer an effective means to attain improved transformation efficiency and ultimately beneficial for the industrial scale in vitro production of the secondary metabolite.

Determination of some conditions for hairy root induction in soybean by Agrobacterium rhizogenes

This study was conducted to determine some conditions for transformation via Agrobacterium rhizogenes on soybean cultivars HLDN29 and DT84. Cotyledon explants were more efficient in hairy root induction compared with hypocotyl explants in both cultivars of soybean. The highest root induction rate and average root number were observed in HLDN29 explants infected with ATCC11325 and ATCC15834 strains (approx. 96% - 100% and 8 roots per explant) and in DT84 explants infected with ATCC15834. Six to eight day - old cotyledonary leaves after sowing were optimal and appropriate for hairy root induction. Direct inoculation and immersion methods showed no significant difference in root induction rate and average root number in both HLDN29 and DT84 cultivars. Transgenic root lines were identified based on PCR analysis with virD and rolC sequences - specific primers.