scholarly journals An Agrobacterium rhizogenes mediated hairy root transformation protocol for fenugreek

MethodsX ◽  
2020 ◽  
Vol 7 ◽  
pp. 101098
Author(s):  
Constantine Garagounis ◽  
Maria-Eleni Georgopoulou ◽  
Konstantina Beritza ◽  
Kalliope K. Papadopoulou
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Transformation Protocol ◽  
Root Transformation

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 Establishment of Agrobacterium rhizogenes-mediated hairy root transformation of Crocus sativus L.

3 Biotech ◽  
2021 ◽  
Vol 11 (2) ◽  
Author(s):  
Shilpi Sharma ◽  
Yeshveer Singh ◽  
Praveen K. Verma ◽  
Jyoti Vakhlu
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Crocus Sativus ◽  
Root Transformation ◽  
Crocus Sativus L

scholarly journals A hairy-root transformation protocol for Trigonella foenum-graecum L. as a tool for metabolic engineering and specialised metabolite pathway elucidation

2020 ◽  
Vol 154 ◽  
pp. 451-462
Author(s):  
Constantine Garagounis ◽  
Konstantina Beritza ◽  
Maria-Eleni Georgopoulou ◽  
Prashant Sonawane ◽  
Kosmas Haralampidis ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Hairy Root ◽  
Transformation Protocol ◽  
Trigonella Foenum Graecum ◽  
Root Transformation ◽  
Trigonella Foenum Graecum L

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 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.

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.

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