Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of Brassica carinata

Hairy roots were produced on faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) plants by inoculation with Agrobacterium root-inducing strains. Examination of 14 plant genotypes and eight Agrobacterium strains in all possible combinations revealed specific strain/genotype interactions. Hairy root size and morphology differed substantially between faba bean and chickpea hairy roots. Sixty percent of chickpea hairy roots were 10–15 mm in length and forty percent, 15–25 mm. All were <1.0 mm in thickness. Sixty-three percent of faba bean hairy roots were 15–25 mm long and thirty-seven percent, 25–40 mm. All faba bean hairy roots were between 1.0 and 1.5 mm in thickness.

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An Efficient Root Transformation System for Recalcitrant Vicia sativa

Frontiers in Plant Science ◽

10.3389/fpls.2021.781014 ◽

2022 ◽

Vol 12 ◽

Author(s):

Vy Nguyen ◽

Iain R. Searle

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Functional Gene ◽

Gene Analysis ◽

Vicia Sativa ◽

Transformation System ◽

Common Vetch ◽

Root Transformation ◽

Functional Gene Analysis

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.

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The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation

BMC Plant Biology ◽

10.1186/s12870-020-02614-x ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Hongmei Shu ◽

Ziliang Luo ◽

Ze Peng ◽

Jianping Wang

Keyword(s):

Hairy Root ◽

Hairy Roots ◽

Root Nodules ◽

Crop Improvement ◽

Functional Study ◽

Nodule Formation ◽

Transformation System ◽

Knock Out ◽

Root Transformation ◽

Peanut Hairy Root

Abstract Background Peanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes. Results We firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut (Tifrunner). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation. Conclusions This study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

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Efficient Genome Editing in Potato Using a Hairy Root Transformation System

Springer Protocols Handbooks - CRISPR-Cas Methods ◽

10.1007/978-1-0716-1657-4_11 ◽

2021 ◽

pp. 149-158

Author(s):

Nathaniel M. Butler ◽

Jiming Jiang

Keyword(s):

Genome Editing ◽

Hairy Root ◽

Transformation System ◽

Root Transformation

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Development of an efficient Agrobacterium -mediated transformation system for Brassica carinata

Plant Cell Reports ◽

10.1007/s002990050375 ◽

1998 ◽

Vol 17 (3) ◽

pp. 183-188 ◽

Cited By ~ 47

Author(s):

V. Babic ◽

R. S. Datla ◽

G. J. Scoles ◽

W. A. Keller

Keyword(s):

Brassica Carinata ◽

Transformation System ◽

Agrobacterium Mediated Transformation

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Root isoflavonoids and hairy root transformation influence key bacterial taxa in the soybean rhizosphere

Environmental Microbiology ◽

10.1111/1462-2920.13602 ◽

2017 ◽

Vol 19 (4) ◽

pp. 1391-1406 ◽

Cited By ~ 13

Author(s):

Laura J. White ◽

Xijin Ge ◽

Volker S. Brözel ◽

Senthil Subramanian

Keyword(s):

Hairy Root ◽

Root Transformation

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First‐generation genome editing in potato using hairy root transformation

Plant Biotechnology Journal ◽

10.1111/pbi.13376 ◽

2020 ◽

Vol 18 (11) ◽

pp. 2201-2209 ◽

Cited By ~ 1

Author(s):

Nathaniel M. Butler ◽

Shelley H. Jansky ◽

Jiming Jiang

Keyword(s):

Genome Editing ◽

Hairy Root ◽

First Generation ◽

Root Transformation

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Hairy Root Transformation in Lotus japonicus

BIO-PROTOCOL ◽

10.21769/bioprotoc.795 ◽

2013 ◽

Vol 3 (12) ◽

Cited By ~ 7

Author(s):

Satoru Okamoto ◽

Emiko Yoro ◽

Takuya Suzaki ◽

Masayoshi Kawaguchi

Keyword(s):

Hairy Root ◽

Lotus Japonicus ◽

Root Transformation

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Detection of Target Sites Edited in Upland Cotton By The CRISPR/Cas9 System Mediated By Agrobacterium Rhizogenes

10.21203/rs.3.rs-1105254/v1 ◽

2021 ◽

Author(s):

Lili Zhou ◽

Yali Wang ◽

Peilin Wang ◽

Jiamin Wang ◽

Hongmei Cheng

Keyword(s):

Agrobacterium Rhizogenes ◽

Hairy Root ◽

Hairy Roots ◽

Upland Cotton ◽

Gene Editing ◽

Target Site ◽

Shoot Meristem ◽

Multiple Target ◽

Root Transformation ◽

Target Sites

Abstract Background CRIPSR/Cas9 gene editing has the ability to effectively modify plant genomes. Multiple target sites usually were designed and the effective target sites were selected for editing. However, upland cotton is allotetraploid and is commonly considered as difficult and inefficient to transform. Therefore, it’s important to quickly identify feasibility of the target site. In this study, we use Agrobacterium rhizogenes K599 strain to infect cotton shoot meristem and induce them to grow hairy roots to detect the feasibility of a selected target designed in GhMYB25-like gene. Results We designed a sgRNA within the second exons of GhMYB25-likeA and GhMYB25-likeD and constructed the CRISPR vector. Transient hairy root transformation using A. rhizogenes K599 with four OD600s (0.4, 0.6,0.8, 1.0) was performed in Coker 312 (R15). The results show that A. rhizogenes at OD600 = 0.6–0.8 is the best concentration range for inducing cotton hairy roots. The other three cultivars (TM-1, Lumian 21, Zhongmian 49) were injected using A. rhizogenes K599 with OD600 = 0.6-0.8 and all produced hairy roots. We characterized ten R15 plants with hairy roots and detected different degrees of base deletions and insert at the target site in five R15 plants. Conclusion Overall, our data show A. rhizogenes-mediated transient hairy root transformation offers a rapid and efficient method to detect sgRNA feasibility in cotton.

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Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean

Plant Methods ◽

10.1186/s13007-021-00778-7 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Yuanyuan Cheng ◽

Xiaoli Wang ◽

Li Cao ◽

Jing Ji ◽

Tengfei Liu ◽

...

Keyword(s):

Functional Analysis ◽

Subcellular Localization ◽

Root System ◽

Hairy Root ◽

Protein Interactions ◽

Gene Editing ◽

Transformation Frequency ◽

Highly Efficient ◽

Root Transformation ◽

Gene Functional Analysis

Abstract Background Agrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function. Results In this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58–64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg’ B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein–protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean. Conclusion A rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein–protein interactions and gene editing system evaluation.

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