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

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.

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.

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.

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.

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 An efficient root transformation system for recalcitrant Vicia sativa

2020 ◽  
Author(s):  
Vy Nguyen ◽  
Iain R. Searle
Keyword(s):  
Hairy Roots ◽  
Functional Gene ◽  
Gene Analysis ◽  
Vicia Sativa ◽  
Transformation System ◽  
Common Vetch ◽  
Root Transformation ◽  
Functional Gene Analysis

AbstractCommon 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 an efficient and rapid hairy root transformation system for common vetch to facilitate functional gene analysis. We infected the hypocotyls of five-day old in vitro or in vivo, soil grown, seedlings with Agrobacterium rhizogenes and produced transformed hairy roots 28 days later at 24% and 43% efficiency, respectively. Seventy-nine percent of the hairy roots from the in vitro plants showed stable expression of a co-transformed marker β-glucuronidase (GUS). In summary, transgenic hairy roots were obtained within 28 days, and are sufficient to facilitate functional gene analysis in common vetch.

scholarly journals A toolbox for nodule development studies in chickpea: a hairy-root transformation protocol and an efficient laboratory strain of Mesorhizobium sp

2018 ◽  
Author(s):  
Drishti Mandal ◽  
Senjuti Sinharoy
Keyword(s):  
Nitrogen Fixation ◽  
Hairy Root ◽  
Root Nodules ◽  
Laboratory Strain ◽  
Nodule Development ◽  
Model Legume ◽  
Transformation Protocol ◽  
Evolutionary Diversification ◽  
Fluorescent Markers ◽  
Root Transformation

AbstractMesorhizobium sp. produces root nodules in chickpea. Chickpea and model legume Medicago truncatula are members of inverted repeat lacking clade (IRLC). The rhizobia after internalization inside plant cell called ‘bacteroid’. Nodule Specific Cysteine-rich (NCR) peptides in IRLC legumes guide bacteroids to a ‘terminally differentiated swollen (TDS)’ form. Bacteroids in chickpea are less TDS than those in Medicago. Nodule development in chickpea indicates recent evolutionary diversification and merits further study. A hairy root transformation protocol and an efficient laboratory strain are prerequisites for performing any genetic study on nodulation. We have standardized a protocol for composite plant generation in chickpea with a transformation frequency above 50%, as shown by fluorescent markers. This protocol also works well in different ecotypes of chickpea. Localization of subcellular markers in these transformed roots is similar to Medicago. When checked inside transformed nodules, peroxisomes were concentrated along the periphery of the nodules, while ER and golgi bodies surrounded the symbiosomes. Different Mesorhizobium strains were evaluated for their ability to initiate nodule development, and efficiency of nitrogen fixation. Inoculation with different strains resulted in different shapes of TDS bacteroids with variable nitrogen fixation. Our study provides a toolbox to study nodule development in the crop legume chickpea.

scholarly journals Agrobacterium rhizogenes-mediated hairy root transformation as an efficient system for gene function analysis in Litchi chinensis

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yaqi Qin ◽  
Dan Wang ◽  
Jiaxin Fu ◽  
Zhike Zhang ◽  
Yonghua Qin ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Gene Function ◽  
Function Analysis ◽  
Transformation System ◽  
Litchi Chinensis ◽  
Gene Function Analysis ◽  
Stem Segments

Abstract Background Litchi chinensis Sonn. is an economically important fruit tree in tropical and subtropical regions. However, litchi functional genomics is severely hindered due to its recalcitrance to regeneration and stable transformation. Agrobacterium rhizogenes-mediated hairy root transgenic system provide an alternative to study functional genomics in woody plants. However, the hairy root transgenic system has not been established in litchi. Results In this study, we report a rapid and highly efficient A. rhizogenes-mediated co-transformation system in L. chinensis using Green Fluorescent Protein (GFP) gene as a marker. Both leaf discs and stem segments of L. chinensis cv. ‘Fenhongguiwei’ seedlings were able to induce transgenic hairy roots. The optimal procedure involved the use of stem segments as explants, infection by A. rhizogenes strain MSU440 at optical density (OD600) of 0.7 for 10 min and co-cultivation for 3 days, with a co-transformation efficiency of 9.33%. Furthermore, the hairy root transgenic system was successfully used to validate the function of the key anthocyanin regulatory gene LcMYB1 in litchi. Over-expression of LcMYB1 produced red hairy roots, which accumulated higher contents of anthocyanins, proanthocyanins, and flavonols. Additionally, the genes involving in the flavonoid pathway were strongly activated in the red hairy roots. Conclusion We first established a rapid and efficient transformation system for the study of gene function in hairy roots of litchi using A. rhizogenes strain MSU440 by optimizing parameters. This hairy root transgenic system was effective for gene function analysis in litchi using the key anthocyanin regulator gene LcMYB1 as an example.

scholarly journals A Toolbox for Nodule Development Studies in Chickpea: A Hairy-Root Transformation Protocol and an Efficient Laboratory Strain of Mesorhizobium sp.

2019 ◽  
Vol 32 (4) ◽  
pp. 367-378 ◽  
Author(s):  
Drishti Mandal ◽  
Senjuti Sinharoy
Keyword(s):  
Nitrogen Fixation ◽  
Hairy Root ◽  
Root Nodules ◽  
Laboratory Strain ◽  
Nodule Development ◽  
Model Legume ◽  
Transformation Protocol ◽  
Evolutionary Diversification ◽  
Fluorescent Markers ◽  
Root Transformation

A Mesorhizobium sp. produces root nodules in chickpea. Chickpea and model legume Medicago truncatula are members of the inverted repeat–lacking clade (IRLC). The rhizobia, after internalization into the plant cell, are called bacteroids. Nodule-specific cysteine-rich peptides in IRLC legumes guide bacteroids to a terminally differentiated swollen (TDS) form. Bacteroids in chickpea are less TDS than those in Medicago spp. Nodule development in chickpea indicates recent evolutionary diversification and merits further study. A hairy-root transformation protocol and an efficient laboratory strain are prerequisites for performing any genetic study on nodulation. We have standardized a protocol for composite plant generation in chickpea with a transformation frequency above 50%, as shown by fluorescent markers. This protocol also works well in different ecotypes of chickpea. Localization of subcellular markers in these transformed roots is similar to the localization observed in transformed Medicago roots. When checked inside transformed nodules, peroxisomes were concentrated along the periphery of the nodules, while endoplasmic reticulum and Golgi bodies surrounded the symbiosomes. Different Mesorhizobium strains were evaluated for their ability to initiate nodule development and efficiency of nitrogen fixation. Inoculation with different strains resulted in different shapes of TDS bacteroids with variable nitrogen fixation. Our study provides a toolbox to study nodule development in the crop legume chickpea.

Induction of Gentiana cruciata hairy roots and their secondary metabolites

Biologia ◽  
2011 ◽  
Vol 66 (4) ◽  
Author(s):  
Sadiye Hayta ◽  
Aynur Gurel ◽  
Ismail Akgun ◽  
Filiz Altan ◽  
Markus Ganzera ◽  
...  
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Root Formation ◽  
Leaf Explants ◽  
Stem Explants ◽  
Root Transformation ◽  
Wild Strains ◽  
Quantitative Results ◽  
Hairy Root Clones ◽  
Gentiana Cruciata

AbstractGentiana cruciata L. (cross gentian) is a medicinal and ornamental plant. The root extracts of this species are known to exhibit many curative properties. The natural Gentiana populations are exposed to great danger because of their uncontrolled usage. In this study, hairy roots from Gentiana cruciata L. stem and leaf explants belonging to three different clones were induced by inoculation with four different Agrobacterium rhizogenes wild strains namely A4, 15834, 8196 and R1000. Induction of the root transformation was significantly dependent on the explant type used. On the other hand, the genotype and bacterial strain had no significant effect on hairy root formation. Hairy root formation percentages of the explants varied between 5.6–33.3% in the stem explants, and between 0.0–6.7% in the leaf explants. Transformations of the hairy roots were confirmed by PCR using rolC specific primers, and revealed the absence of contaminating A. rhizogenes with virC primers. Total of twelve hairy root clones were obtained, and their secondary metabolite content was also analyzed by HPLC. Quantitative results exhibited that gentiopicroside was the most abundant compound in all root samples. Furthermore, metabolites such as loganic acid, swertiamarin, and sweroside were also identified and quantified in the samples.

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