Hairy Root Transformation in Lotus japonicus

BIO-PROTOCOL ◽  
2013 ◽  
Vol 3 (12) ◽  
Author(s):  
Satoru Okamoto ◽  
Emiko Yoro ◽  
Takuya Suzaki ◽  
Masayoshi Kawaguchi
Keyword(s):  
Hairy Root ◽  
Lotus Japonicus ◽  
Root Transformation

scholarly journals Use of the rhizobial type III effector gene nopP to improve Agrobacterium rhizogenes-mediated transformation of Lotus japonicus

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Wang ◽  
Feng Yang ◽  
Peng-Fei Zhu ◽  
Asaf Khan ◽  
Zhi-Ping Xie ◽  
...  
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Fluorescent Protein ◽  
Transformation Efficiency ◽  
Lotus Japonicus ◽  
Nodule Bacterium ◽  
Transgenic Roots ◽  
Model Legume ◽  
Effector Gene ◽  
Root Transformation

Abstract Background Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. Conclusion We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

scholarly journals Gene Silencing by Expression of Hairpin RNA in Lotus japonicus Roots and Root Nodules

2003 ◽  
Vol 16 (8) ◽  
pp. 663-668 ◽  
Author(s):  
Hirotaka Kumagai ◽  
Hiroshi Kouchi
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Rna Silencing ◽  
Root Nodules ◽  
Lotus Japonicus ◽  
Loss Of Function ◽  
Coding Region ◽  
Hairpin Rna ◽  
Model Legume ◽  
Root Transformation

We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes. Transgenic lines that express β-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A. rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region. GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely. Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis. These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.

HAIRY ROOT TRANSFORMATION SYSTEM IN LARGE-SEEDED GRAIN LEGUMES

1995 ◽  
Vol 43 (1) ◽  
pp. 1-5 ◽  
Author(s):  
H.J. Siefkes-Boer ◽  
M.J. Noonan ◽  
D.W. Bullock ◽  
A.J. Conner
Keyword(s):  
Vicia Faba ◽  
Hairy Root ◽  
Hairy Roots ◽  
Faba Bean ◽  
Grain Legumes ◽  
Transformation System ◽  
Root Transformation ◽  
Specific Strain ◽  
Size And Morphology ◽  
Root Size

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.

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

2017 ◽  
Vol 19 (4) ◽  
pp. 1391-1406 ◽  
Author(s):  
Laura J. White ◽  
Xijin Ge ◽  
Volker S. Brözel ◽  
Senthil Subramanian
Keyword(s):  
Hairy Root ◽  
Root Transformation

scholarly journals First‐generation genome editing in potato using hairy root transformation

2020 ◽  
Vol 18 (11) ◽  
pp. 2201-2209 ◽  
Author(s):  
Nathaniel M. Butler ◽  
Shelley H. Jansky ◽  
Jiming Jiang
Keyword(s):  
Genome Editing ◽  
Hairy Root ◽  
First Generation ◽  
Root Transformation

scholarly journals An Efficient Root Transformation System for Recalcitrant Vicia sativa

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.

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

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.

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

Plant Methods ◽  
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.

scholarly journals Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

Plants ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 51
Author(s):  
Alexey S. Kiryushkin ◽  
Elena L. Ilina ◽  
Elizaveta D. Guseva ◽  
Katharina Pawlowski ◽  
Kirill N. Demchenko
Keyword(s):  
Genome Editing ◽  
Hairy Root ◽  
Fluorescent Protein ◽  
Effective Means ◽  
Gene Encoding ◽  
Transgenic Roots ◽  
Translational Enhancer ◽  
Current State ◽  
Root Transformation ◽  
Agrobacterium Rhizogenes Strains

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

scholarly journals GmVTL1 is an iron transporter on the symbiosome membrane of soybean with an important role in nitrogen fixation

2020 ◽  
Author(s):  
Ella M. Brear ◽  
Frank Bedon ◽  
Aleksandr Gavrin ◽  
Igor S. Kryvoruchko ◽  
Ivone Torres-Jerez ◽  
...  
Keyword(s):  
Lotus Japonicus ◽  
List Type ◽  
Symbiotic Relationships ◽  
Iron Transporter ◽  
Symbiosome Membrane ◽  
Transporter Family ◽  
Root Transformation ◽  
Carbon Substrates ◽  
Infected Cells

SummaryLegumes establish symbiotic relationships with soil bacteria (rhizobia), housed in nodules on plant roots. The plant supplies carbon substrates and other nutrients to the bacteria in exchange for fixed nitrogen. The exchange occurs across a plant-derived symbiosome membrane (SM), which encloses rhizobia to form a symbiosome. Iron supplied by the plant is crucial for the rhizobial enzyme nitrogenase that catalyses N2 fixation, but the SM iron transporter has not been identified.We use complementation of yeast and plant mutants, real-time PCR, hairy root transformation, microscopy and proteomics to demonstrate the role of soybean GmVTL1 and 2.Both are members of the vacuolar iron transporter family and homologous to Lotus japonicus SEN1 (LjSEN1), previously shown to be essential for N2 fixation. GmVTL1 expression is enhanced in nodule infected cells and both proteins are localised to the SM.GmVTL1 and 2 transport iron in yeast and GmVTL1 restores N2 fixation when expressed in the Ljsen1 mutant.Three GmVTL1 amino acid substitutions that reduce iron transport in yeast also block N2 fixation in Ljsen1 plants.We conclude GmVTL1 is responsible for transport of iron across the SM to bacteroids and plays a crucial role in the N2-fixing symbiosis.

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